Progress 12/01/09 to 11/30/14
Outputs
Target Audience: The target audience for the published work is other research investigators who work in the areas of DNA replication and DNA repair, or in related areas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Drs. Chodavarapu and Felczak are postdoctoral students who conducted the experiments in the article by Chodavarapu et al. Dr. Simmons and Mr. Murillo were graduate students. The work leading to this publication is part of their training and professional development. How have the results been disseminated to communities of interest? The results by Chodavarapu et al. have been published in Nucleic Acids Research, an "open access" journal. The review articles may be obtained by requesting them via email, or by subscription. What do you plan to do during the next reporting period to accomplish the goals? In the next funding period, we will pursue several major aims. In Aim 1, we will determine if the binding of DnaC to DnaB affects the conformation of the N-terminal domain of DnaB so that primase cannot interact with the helicase. This set of experiments tests the hypothesis that DnaC acts as a molecular switch that controls whether DnaB can interact with primase or not. The second aim will identify regions within the N-terminal domain of DnaA that interact with other proteins to influence the helicase loading process. The hypothesis underlying this aim is that these proteins affect the activity of DnaA to modulate the frequency of initiation in response to different in vivo conditions. A third aim relies on cryoelectron microscopic characterization of DnaA, DnaB and DnaC assembled at a DnaA box sequence. The purpose of this aim is to understand the molecular architecture of the DnaA-DnaB-DnaC complex in order to relate their structures to how these proteins function. These studies will greatly advance our understanding of the mechanism and regulation of helicase recruitment.
Impacts
What was accomplished under these goals?
The focus of our laboratory is to understand the biochemistry of replication initiation, and how this process is regulated to ensure that it occurs only once per cell cycle. The publication by Chodavarapu et al. describes the isolation and characterization of mutant DnaAs that overinitiate DNA replication by virtue of their relative inability to respond to regulatory mechanisms that control initiation. Of particular interest, the properties of one mutant suggest that it fails to respond to a novel regulatory factor. Other manuscripts that are in press will appear as book chapters that review recent findings in the field of DNA replication. These book chapters are helpful summaries for scientists who study DNA replication, and for others who work in related areas. Manuscripts that are in preparation describe the interaction of DnaC with DnaB, and that this interaction traps DnaB in a conformation whereby adjacent DnaB protomers of the DnaB toroid are separated to form a gap in the DnaB ring. This conformation of DnaB occludes a site in the DNA helicase so that primase cannot interact. These and our previous studies show that DnaC controls the function of DnaB in DNA replication. The gap described above serves as the entry site through which the single-stranded DNA will pass in order to bind to the interior of the DnaB ring. After DNA binding, DnaB can then act as a DNA helicase to unwind duplex DNA.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Chodavarapu, S., Felczak, M.M., Simmons, L.A., Murillo, A., and Kaguni, J.M. (2013) Mutant DnaAs of Escherichia coli that are refractory to negative control. Nucleic Acids Res. 41, 10254-10267 PMID: 23990329
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2014
Citation:
Kaguni, J.M. DNA replication. The Encyclopedia of Molecular Life Sciences. R.D. Wells, J. S. Bond, J.P. Klinman, B. S.S. Masters, and J.E. Bell, eds. Elsevier Science. in press
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2014
Citation:
Kaguni, J.M. DnaA, DnaB and DnaC. The Encyclopedia of Molecular Life Sciences. R.D. Wells, J. S. Bond, J.P. Klinman, B. S.S. Masters, and J.E. Bell, eds. Elsevier Science. in press
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2014
Citation:
Kaguni, J.M. The replication origin of E. coli and the mechanism of initiation. The Encyclopedia of Molecular Life Sciences. R.D. Wells, J. S. Bond, J.P. Klinman, B. S.S. Masters, and J.E. Bell, eds. Elsevier Science. in press
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2014
Citation:
Kaguni, J.M. Control of initiation in E. coli. The Encyclopedia of Molecular Life Sciences. R.D. Wells, J. S. Bond, J.P. Klinman, B. S.S. Masters, and J.E. Bell, eds. Elsevier Science. in press
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience for the published work is other research investigators who work in the areas of DNA replication and DNA repair, or in related areas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Drs. Chodavarapu and Felczak are postdoctoral students who conducted the experiments in the article by Chodavarapu et al. Dr. Simmons and Mr. Murillo were graduate students. The work leading to this publication is part of their training and professional development. How have the results been disseminated to communities of interest? The results by Chodavarapu et al. have been published in Nucleic Acids Research, an "open access" journal. The review articles may be obtained by requesting them via email, or by subscription. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A major objective of this work is to understand how DNA replication is regulated so that it occurs only once per cell cycle. The publication by Chodavarapu et al. describes the isolation and characterization of mutant DnaAs that overinitiate DNA replication by virtue of their relative inability to respond to regulatory mechanisms that control initiation. Of particular interest, the properties of one mutant suggest that it fails to respond to a novel regulatory factor. Other publications are book chapters that summarize recent developments in the field of DNA replication. These up-to-date reviews are helpful to research investigators working in the area of DNA replication, and to others performing research in related areas.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Chodavarapu, S., Felczak, M.M., Simmons, L.A., Murillo, A., and Kaguni, J.M. (2013) Mutant DnaAs of Escherichia coli that are refractory to negative control. Nucleic Acids Res. PMID: 23990329
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Kaguni, J.M. DNA Replication: Initiation in Bacteria. (2013) The Encyclopedia of Biological Chemistry, 2e. W.J. Lennarz and M.D. Lane, eds. Elsevier Science pp. 761-766
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2013
Citation:
Bell, S.P. and Kaguni, J.M., Helicase Loading at Chromosomal Origins of Replication. (2013) In DNA Replication, S.P. Bell, M. M�chali and M. DePamphilis, eds. Cold Spring Harbor Press.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The broad objectives of this project are to understand the mechanism of initiation of chromosomal DNA replication and its regulation in E. coli. These studies using this model organism should lead to new insight into how these processes occur in higher organisms. This expectation is based on evidence showing that these processes are biochemically similar among all free-living organisms. In the last funding period, we reported that an increased abundance of DnaA induces hyper-initiation, which leads to more double strand breaks in DNA. Apparently, these breaks arise when recently formed replication forks collide from behind with stalled or collapsed forks. In mutants that are defective in DNA repair, these breaks are toxic. Using a multicopy suppressor assay to select genes that neutralize this toxicity, we identified a gene whose function had been unknown. Our work shows that this gene reduces the frequency of chromosome breaks. By lowering the steady-state level of double strand breaks, it helps to maintain the integrity of the bacterial chromosome. We propose that this gene confers a selective growth advantage to E. coli K12 strains. PARTICIPANTS: Dr. Magdalena M. Felczak is a postdoctoral fellow who conducted the experiments in the relevant publication. The published work represents part of her training and professional development. TARGET AUDIENCES: The target audiences for the publications are other research investigators that work in the areas of DNA replication, and DNA repair, as well as the larger scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In all free-living organisms, chromosomal DNA replication is essential for viability, initiating at a specific time during the cell cycle. Our major aims are to investigate the molecular mechanisms of initiation of DNA replication and its regulation using E. coli as a model organism. A major focus is on the biochemistry of proteins required at the stage of initiation. Recently, we reported on the characterization of a gene whose function was unknown. We discovered that it is involved in maintaining the integrity of the E. coli chromosome; the absence of this gene leads to an increased abundance of broken chromosomal DNA. We suggest that the presence of this gene in E. coli provides a growth advantage. Further work on how cells maintain the integrity of its genome may lead to advances in agriculture and medicine.
Publications
- Felczak, M.M., and Kaguni, J.M. 2012. The rcbA gene product reduces spontaneous and induced chromosome breaks in Escherichia coli. J. Bacteriol. 194: 2152-2164.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The long-term goals of this project are to understand the mechanism of initiation of chromosomal DNA replication and its regulation in E. coli. Studies of this model organism by independent laboratories over several decades demonstrate that these processes are very similar to those occurring in more complex organisms, so the objectives have broad relevance. Earlier work from our lab showed that elevated levels of DnaA cause excessive initiation, which leads to an increased level of double strand breaks that are proposed arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that neutralize this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. In a manuscript under review, this gene encoded by the cryptic rac prophage has been named rcbA for its ability to reduce the frequency of chromosome breaks. Our study shows that colony formation of strains bearing mutations in rep, recG and rcbA, like recA and recB mutants, is inhibited by oversupply of DnaA, and that a multicopy plasmid carrying rcbA suppresses this inhibition. These and other results suggest that rcbA helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double strand breaks. Hence, rcbA provides a selective growth advantage to E. coli K12 strains that have the rac prophage in the genome. We also reported that ribosomal protein L2 and a truncated form inhibit DnaA function in vitro. Because DNA replication is coordinated with cell growth, which is largely determined by the rate of ribosome biogenesis, we suggest that surplus L2 that may accumulate when the levels of components required for ribosome biogenesis become disproportionate may inhibit DnaA function to modulate the initiation process, and to couple the initiation of DNA replication with cell growth. PARTICIPANTS: Drs. Sundari Chodavarapu, and Magdalena M. Felczak are postdoctoral fellows who conducted the experiments in the relevant publications. The published work represents part of their training and professional development. TARGET AUDIENCES: The target audiences for the publications are other research investigators that work in the areas of DNA replication, and protein synthesis, as well as the larger scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Chromosomal DNA replication is an essential process. In the bacterial cell cycle of E. coli, chromosomal DNA replication initiates at a specific time due to the function of DnaA protein. The long-term objectives of this work are to investigate the molecular mechanisms of initiation of DNA replication and its regulation. Because DnaA protein regulates the frequency of initiation, a major focus has been on its biochemical characterization. The discoveries from this work may lead to advances in agriculture and medicine. In the last funding period, we isolated a gene of unknown function and showed that it apparently reduces the steady-state level of double strand breaks in E. coli chromosomal DNA. We suggest that the presence of this gene in some E. coli strains provides a growth advantage. In a separate study, we showed that ribosomal protein L2 and a truncated form physically interacts with the N-terminal region of DnaA to inhibit the initiation of DNA replication. Specific biochemical experiments reveal that either form of L2 interferes with DnaA oligomer formation, the unwinding of the E. coli replication origin, and the subsequent assembly of an intermediate named the prepriming complex that assembles at the E. coli replication origin. These observations suggest that one or both forms of L2 may modulate DnaA function in vivo to regulate the frequency of initiation.
Publications
- Kaguni, J.M. (2011) Replication initiation at the Escherichia coli chromosomal origin. Current Opinion in Chemical Biology. S. Benkovic and K.D. Raney, eds. Elsevier. 15:1-8
- Hupert-Kocurek, K., and Kaguni, J.M. (2011) Understanding protein function, the disparity between bioinformatics and molecular methods. Computational Biology and Applied Bioinformatics. Heitor Silverio Lopes and Leonardo Magalhes Cruz (Ed.), ISBN: 978-953-307-629-4 InTech, Available from: http://www.intechopen.com/articles/show/title/understanding-protein-f unction-the-disparity-between-bioinformatics-nd-molecular-methods
- Kaguni, J.M. (2011) DNA Replication: Initiation in Bacteria. The Encyclopedia of Biological Chemistry, 2e. W.J. Lennarz and M.D. Lane, eds. Elsevier Science pp. 761-766
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This project investigates the mechanism of initiation of chromosomal DNA replication in E. coli. Much of our understanding of this process has been obtained from this model organism, which serves as a benchmark to understand initiation in higher life forms. The accumulated evidence from a number of independent investigators reveals that this process is very similar in all free-living organisms. We report on progress in two areas during the past year. One of the previous objectives was to determine the mechanism of inhibition of initiation by Dps, a protein that protects the bacterial chromosome from oxidative damage. During these studies, we detected another protein that inhibited chromosomal DNA replication in vitro, purified it, and identified it by a variety of methods as a truncated form of ribosomal protein L2. As one of the most evolutionarily ancient among ribosomal proteins, it is essential for ribosome biogenesis. Because ribosome assembly and DNA replication are coupled with cell growth, we considered the possibility that L2 may inhibit DnaA function to modulate the initiation process, and to coordinate the initiation of DNA replication with cell growth. These studies are reported in an article that is in press. A second objective investigates the role of E. coli DnaC protein during initiation. DnaC forms a complex with DnaB helicase, and this complex is required for this helicase to bind to the E. coli chromosomal origin. However, the mechanism whereby DnaC delivers DnaB helicase at the chromosomal origin is poorly understood. DnaC also binds ATP, but the importance of ATP binding and ATP hydrolysis are uncertain. We asked if DnaC must bind ATP to deliver DnaB to oriC, and if ATP hydrolysis leads to the dissociation of DnaC from DnaB. Experiments that address these questions are described in a published report. PARTICIPANTS: Drs. Magdalena Makowska-Grzyska, Sundari Chodavarapu, and Magdalena M. Felczak are postdoctoral fellows who conducted most of the experiments in the publications. The published work represents part of their training and professional development. TARGET AUDIENCES: The target audiences for the publications are other research investigators that work in the areas of DNA replication, and protein synthesis, as well as the larger scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, chromosomal replication is strictly regulated to occur at a specific time in the bacterial cell cycle. DnaA protein functions at initiation to regulate the frequency of initiation. This work, which may have applications to the fields of medicine and agriculture, investigates the molecular mechanisms of initiation of DNA replication and its regulation. As part of one objective, we showed that a truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation. Fulfilling a second objective that investigates the role of DnaC in initiation, we showed that mutant DnaC proteins bearing alanine substitutions for two conserved arginines in a motif named box VII are defective in DNA replication, but this deficiency does not arise from impaired interactions with ATP, DnaB or single-stranded DNA. Despite their ability to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive. Quantitative analysis of the prepriming complex suggests that the DnaB-DnaC complex contains three DnaC monomers per DnaB hexamer, and that the interaction of primase with DnaB and primer formation triggers the release of DnaC but not the mutants from DnaB. The interaction of primase with DnaB, and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.
Publications
- Makowska-Grzyska, M. and Kaguni, J.M. 2010. Primase directs the release of DnaC from DnaB. Mol. Cell. 37:90-101.
- Chodavarapu, S., Felczak, M.M. and Kaguni, J.M. 2010. Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication. Nucleic Acids Res. In press.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: DnaAcos hyperinitiates by circumventing regulatory pathways that control the frequency of initiation in Escherichia coli - Previously, we showed that elevated levels of DnaA protein induce extra initiations by apparently overcoming regulatory pathways that control the frequency of initiation of DNA replication in E. coli. Because the extra initiations cause lethality in strains unable to repair double-strand breaks, supporting a model that newly formed replication forks collide from behind with stalled forks followed by fork collapse. To identify genes and loci that regulate the frequency of initiation, we developed a genetic method to select for plasmids carrying E. coli DNA fragments that suppress the lethal effect caused by elevated dnaA expression. Via this approach, we identified seqA, datA, dnaN and hda, which respectively are known either to act at oriC, or to modulate the activity of DnaA protein to affect initiation frequency. We then assessed each pathway's relative effectiveness in regulating initiation. These results indicate that the hda-dependent pathway has a stronger effect on initiation frequency than pathways involving seqA and datA. This work has been published in a peer-reviewed journal. Primase Directs the Release of DnaC from DnaB - During initiation, DnaC delivers DnaB helicase at the E. coli chromosomal origin, but this process is poorly understood. In other work, we showed that mutant proteins bearing alanine substitutions for two conserved arginines in a motif named box VII are defective in DNA replication, but this deficiency does not arise from impaired interactions with ATP, DnaB or single-stranded DNA. Despite the ability of these mutant DnaC proteins to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive. Quantitative analysis of the prepriming complex suggests that the DnaB-DnaC complex contains three DnaC monomers per DnaB hexamer, and that the interaction of primase with DnaB and primer formation triggers the release of DnaC but not the mutants from DnaB. The interaction of primase with DnaB, and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.This work has been published in a peer-reviewed journal. Ribosomal protein L2 inhibits the assembly of the prepriming complex - We recently discovered that ribosomal protein L2 inhibits DNA replication of a plasmid containing the E. coli chromosomal origin and the assembly of the oriC prepriming complex by obstructing the unwinding of oriC by DnaA. We also determined that DnaA and L2 physically interact by ELISA. Current experiments show that an elevated level of this ribosomal protein also interferes with the initiation of bacterial DNA replication in vivo. As the expression of ribosomal genes and ribosome biogenesis are tightly coordinated with cell growth, we suggest that when L2 is in excess, it interacts with DnaA to inhibit the initiation of DNA replication, and to couple initiation with ribosome assembly. A manuscript is under revision. PARTICIPANTS: Jon M. Kaguni is the principal investigator of this project. He directed the experiments under each specific aim, analyzed the results, and co-authored the peer-reviewed publications. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, DnaA protein initiates chromosomal DNA replication at a specific time in the bacterial cell cycle by directing the assembly at the replication origin of the enzymatic machinery needed to duplicate the chromosome. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. Results obtained during this funding period provide insight into these processes. Other findings suggest a connection between the initiation of DNA replication and ribosome biogenesis. This work may have applications to the fields of medicine and agriculture.
Publications
- Felczak, M.M., and Kaguni, J.M. 2009. DnaAcos hyperinitiates by circumventing regulatory pathways that control the frequency of initiation in Escherichia coli. Mol Microbiol. 72:1348-1363.
- Makowska-Grzyska, M. and Kaguni, J.M. 2009. Primase Directs the Release of DnaC from DnaB. Mol. Cell (in press).
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Independent pathways regulate the frequency of initiation of the E. coli chromosome - We previously showed that elevated dnaA expression induces extra initiations, which cause lethality in strains defective in the repair of double strand breaks. The results support a model that the newly formed replication forks collide from behind with stalled forks, causing lethal fork collapse. Based on suppression of the lethal effect caused by elevated dnaA expression, we developed a genetic method to select for multicopy suppressors that regulate the frequency of initiation, and obtained plasmids carrying seqA, datA, dnaN and hda, which respectively are known either to act at the replication origin (oriC), or to modulate the activity of DnaA protein. We also used strains genetically inactivated in each pathway to assess their relative effectiveness in regulating initiation when the level of DnaA protein was elevated. Our results indicate that the hda-dependent pathway has a stronger effect on initiation frequency than the pathways involving seqA and datA. Dps regulates DnaA function - During exponential growth, the level of Dps transiently increases in response to oxidative stress to sequester and oxidize iron, which would otherwise lead to hydroxyl radicals that damage the bacterial chromosome. We discovered that Dps specifically interacts with the N-terminal region of DnaA protein by affinity chromatography and a solid phase binding assay. In vitro, we showed that Dps inhibits DnaA function in initiation by interfering with strand opening of oriC. We then used two independent in vivo methods of flow cytometry and quantitative PCR analysis to confirm the in vitro results that Dps reduces initiation frequency. We suggest that Dps reduces initiations to act as a checkpoint during oxidative stress, thereby providing an opportunity for mechanisms to repair oxidative DNA damage. Because Dps does not block initiations absolutely, duplication of the damaged DNA is expected to increase the genetic variation of a population, and the probability that genetic adaptation leads to survival under conditions of oxidative stress. Ribosomal protein L2 inhibits the assembly of the prepriming complex - In experiments to purify Dps for its biochemical study, we detected a protein that inhibited oriC plasmid replication. Based on the protein's inhibitory activity, we purified it and identified it as ribosomal protein L2 by N-terminal sequence analysis. We showed that DnaA and L2 physically interact by ELISA to confirm an independent report, and that L2 inhibits the unwinding of oriC by DnaA and the assembly of the oriC prepriming complex. As the expression of ribosomal genes and ribosome biogenesis are tightly coordinated with cell growth, we suggest that when L2 is in excess, it interacts with DnaA to inhibit the initiation of DNA replication, and to couple initiation with ribosome assembly. PARTICIPANTS: Jon M. Kaguni is the principal investigator who helped design experiments and interpret experimental data. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, chromosomal replication occurs at a specific time in the bacterial cell cycle and is a highly regulated event. DnaA protein functions at initiation to regulate the frequency of initiation. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. This work may have applications to the fields of medicine and agriculture.
Publications
- Chodavarapu, S., Felczak, M. M., Rouviere-Yaniv, J., and Kaguni, J. M. 2008. Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin. Mol Microbiol 67: 781-792.
- Chodavarapu, S., Gomez, R., Vicente, M., and Kaguni, J.M. 2008. Escherichia coli Dps interacts with DnaA protein to impede initiation: a model of adaptive mutation. Mol Microbiol 67: 1331-1346.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: A genetic method to analyze essential genes of Escherichia coli: Because it is difficult to analyze essential genes of E. coli by genetic methods, we developed a genetic method for the mutational analysis using the dnaC gene as an example, and obtained a large collection of novel dnaC mutations. This methodology should be widely applicable to the genetic analysis of other essential genes of E. coli and related enterobacteria. We are currently analyzing several mutant DnaCs to understand the biochemical mechanism of this protein in initiation. Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin: E. coli HU protein is a dimer encoded by two closely related genes whose expression is growth-phase dependent. As a major component of the bacterial nucleoid, HU binds to DNA nonspecifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We showed by affinity chromatography and a solid phase
binding assay that HU interacts via its alpha subunit with DnaA. We also showed that the alpha dimer of HU more effectively promotes strand opening of oriC and is more active than other forms of HU in initiation of an oriC-containing plasmid. Other results suggest that HU stabilizes the DnaA oligomer bound to oriC. These observations support a model whereby DnaA interacts with the alpha dimer or the alpha-beta heterodimer, depending on their cellular abundance, to recruit the respective forms of HU to oriC. The greater activity of the alpha dimer of HU at oriC may stimulate initiation during early log phase growth. Escherichia coli Dps interacts with DnaA to lower initiation frequency, supporting a model that maintains DNA integrity while also permitting adaptive mutagenesis: The results of this study support a model that, in exponentially growing cells subjected to oxidative stress, the interaction of Dps with DnaA acts as a checkpoint to inhibit initiation until oxidative damage to
DNA is repaired. A submitted manuscript is under revision. Independent pathways regulate the frequency of initiation of the Escherichia coli chromosome: Using a genetic approach, we identified genes that regulate the frequency of initiation from oriC. Some of these genes were previously shown to regulate initiation from oriC, confirming the validity of the approach. Other genes are being characterized. A manuscript describing this work is in preparation. Mutant DnaAs that promote excessive initiation at oriC: We developed a genetic method to isolate mutations in dnaA encoding proteins that are defective in responding to regulatory factors that control the frequency of initiation. After isolating a small collection of novel dnaA mutations, we showed that the mutant DnaAs promote excessive initiations, and suggest that they presumably fail to respond to regulatory factors that normally control the frequency of initiation. These proteins are being characterized to understand their
biochemical defects.
PARTICIPANTS: Jon M. Kaguni, Principal Investigator Department of Biochemistry & Molecular Biology Michigan State University Josette Rouviere-Yaniv, collaborator Laboratoire de Physiologie Bacterienne CNRS, UPR 9073 Institut de Biologie Physico-Chimique 13 rue Pierre et Marie Curie Paris, 75005 France
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, chromosomal replication occurs at a specific time in the bacterial cell cycle and is a highly regulated event. DnaA protein functions at initiation to regulate the frequency of initiation. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. This work may have applications to the fields of medicine and agriculture.
Publications
- Hupert-Kocurek, K., Sage, J. M., Makowska-Grzyska, M., and Kaguni, J. M. 2007. A genetic method to analyze essential genes of Escherichia coli. App. Environ. Microbiol. 73: 7075-7082.
- Chodavarapu, S., Felczak, M. M., Rouviere-Yaniv, J., and Kaguni, J. M. 2008. Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin. in press.
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Progress 01/01/06 to 12/31/06
Outputs
Dps modulates DnaA function. Dps is a nucleoid-associated protein that binds and oxidizes Fe2+ to sequester the metal, thus blocking the production of hydroxyl radicals to protect cells from oxidative DNA damage. During exponential growth, oxidative stress leads to induced transcription of the dps gene. In stationary phase cells, Dps as the most abundant DNA binding protein also protects DNA and condenses the chromosome into a biocrystal in starved cells. We show that Dps interacts specifically with DnaA protein by affinity chromatography and a solid phase binding assay. Flow cytometry experiments comparing isogenic dps+ and dps::kan strains suggest that Dps reduces the frequency of initiations. Other results indicate that Dps inhibits initiation by blocking the DnaA-dependent unwinding of the chromosomal origin. These results suggest that Dps may act as a checkpoint during oxidative stress to inhibit initiation until oxidative damage to DNA is repaired, thereby
maintaining the integrity of the bacterial genome. Identification of genes that regulate initiation. Earlier, we showed by quantitative methods that an elevated level of DnaA induces extra initiations. The newly formed replication forks collide from behind with forks that have stalled, leading to fork collapse and double strand breaks. If the cell cannot repair the double strand breaks, the extra initiations lead to cell death. We hypothesized that, because DnaA controls the frequency of initiation from oriC, we should be able to isolate genes contained in a multicopy plasmid that regulate the frequency of initiation by negatively modulating DnaA function. We also expected to identify genes such as seqA whose products act directly at oriC to inhibit extra initiations, and other classes of genes that either slow the rate of replication fork movement to reduce the frequency of collision with newly propagated forks, with a downstream stalled fork, or that minimize or prevent forks from
stalling. A fifth class is genes that enhance the efficiency of double strand break repair. To exclude the last class, we constructed a plasmid library in which we inserted chromosomal DNA fragments form a recA mutant strain into the multicopy plasmid, pACYC184. We developed a genetic method to select recombinant plasmids containing genes that regulate DnaA activity. One set of recombinant plasmids encodes seqA. Presumably, the elevated copy number of the plasmid leads to an increased abundance of SeqA protein and more effective sequestration of oriC when it becomes hemi-methylated after an initiation cycle, thereby inhibiting unscheduled initiations. Another group of plasmids carry either hda or dnaN, which encodes the β subunit of DNA polymerase III holoenzyme. We showed genetically that both genes act in the same pathway to stimulate the hydrolysis of ATP bound to DnaA to convert DnaA from a form that is active in initiation to an inactive form. A third class of plasmids is
those that carry datA, which functions to titrate excess DnaA to reduce initiation frequency. Other genes have been identified and are being characterized to determine how they alleviate the lethal effect of DnaA overproduction.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, chromosomal replication occurs at a specific time in the bacterial cell cycle and is a highly regulated event. DnaA protein functions at initiation to regulate the frequency of initiation. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. This work may have applications to the fields of medicine and agriculture.
Publications
- Kaguni, J. M. 2006. DnaA: Controlling the Initiation of Bacterial DNA Replication and More. Annu. Rev. Microbiol. 60: 351-371.
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Progress 01/01/05 to 12/31/05
Outputs
DnaA oligomerization-Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Of the four domains identified in structure-function studies by our laboratory and others, we previously showed that Domain I near the N-terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC. We asked if these functions are separable or interdependent. We showed that alanine substitutions of leucine 3, phenylalanine 46 and leucine 62 did not affect DnaA function in initiation. In contrast, detailed biochemical characterization of a mutant DnaA showed that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogated self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase. Other results also indicate that DnaA
oligomerization is essential for initiation. By characterizing dnaA alleles suspected from in vivo evidence to be partially active in DNA replication, we showed biochemically that one mutant DnaA was defective in self-oligomerization. Other results from the study of other mutant DnaAs suggest that this function stabilizes the binding of DnaA to the bacterial replication origin. HU modulates DnaA function-We previously described a number of proteins that specifically and reproducibly interact with DnaA as an affinity chromatography ligand. One of these is HU, a small basic protein involved in a number of DNA metabolic reactions. During log phase growth, its composition is a mixture of a homodimer of two alpha subunits and a heterodimer of an alpha and beta subunit encoded by the hupA and hupB genes, respectively. As cells enter stationary phase, the hupB gene is expressed. Because HU is relatively stable, the composition of HU in stationary phase cells is the heterodimer and the
homodimer of two beta subunits. We compared the alpha dimer to the beta dimer of HU and found that the alpha dimer was more active than the beta dimer in oriC plasmid replication at the step of unwinding of oriC. Because HU stimulates initiation at oriC at the step of unwinding, we speculate that the specific interaction between DnaA and the alpha dimer acts to recruit HU to oriC. When cells enter stationary phase, the increased abundance of the beta dimer may result in less efficient initiation from the failure to recruit HU.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms. In E. coli, chromosomal replication occurs at a specific time in the bacterial cell cycle and is a highly regulated event. DnaA protein functions at initiation to regulate the frequency of initiation. This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. This work may have applications to the fields of medicine and agriculture.
Publications
- Felczak, M.M., Simmons, L.A., and Kaguni, J.M. 2005. An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin. J Biol Chem 280: 24627-24633.
- Walker, J. R., Severson, K. A., Hermandson, M. J., Blinkova, A., Carr, K. M., and Kaguni, J. M. 2006. Escherichia coli DnaA protein: Specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation. Biochimie 88: 1-10.
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Progress 01/01/04 to 12/31/04
Outputs
Initiation of DNA replication from the E. coli chromosomal origin, oriC, occurs through a series of discrete biochemical steps. First, DnaA protein binds to DnaA box sequences within oriC, then recruits DnaB helicase to an unwound region within it. DnaB bound to the separated parental DNA strands then translocates on the DNA to further unwind the parental duplex. Primase then synthesizes primers that are extended by DNA polymerase III holoenzyme to duplicate the chromosome. Hyperinitiation causes replication fork collapse and inviability. We studied a dnaA mutation named dnaAcos, which is lethal because it induces extra initiations. In contrast, induced expression of dnaA+ from a multicopy plasmid increases initiation frequency but viability is maintained. By quantitative methods, we showed that the elevated initiations induced by dnaAcos leads to a larger amount of collapsed replication forks, which is the cause for its lethality. We also showed that when the
replication restart pathway is compromised by mutations in genes involved in double strand break repair, the lower level of initiation by induced dnaA+ is now lethal. Thus, inviability caused by either dnaAcos or induced dnaA+ expression is from the failure to repair double strand breaks. Self-aggregation of DnaA. We identified specific amino acids within an N-terminal region of DnaA required for self-aggregation. We then showed biochemically that a mutant DnaA carrying the W6A substitution is inactive in initiation because it fails to self-oligomerize. We conclude that DnaA oligomerization at oriC requires specific amino acids in an N-terminal domain for initiation. The Box VII motif of DnaA. The AAA+ family of ATPases carries a motif named Box VII which bears a conserved arginine (the arginine finger) proposed to sense the hydrolysis of the bound nucleotide in coordinating domain movement during protein function. Arginine 281 of DnaA, an AAA+ family member, may act as the arginine
finger residue. As DnaA oligomerization is essential for initiation, DnaA seems like other AAA+ proteins which assemble into oligomeric structures. Because ATP binding but hot its hydrolysis by DnaA is required to unwind oriC, one prediction is that a mutant in the Box VII motif should bind ATP, and thus be able to unwind oriC. The second is that, because the unwound structure is thought to be sufficient for DnaB to bind, the mutant DnaA should be active in DNA replication from oriC. As a test, we substituted the conserved amino acids in Box VII with alanine, and showed that a representative mutant DnaA (R281A) retains wild type activity in ATP binding and in unwinding oriC. In contrast, R281A and other mutant Box VII proteins are inactive in initiation at oriC, showing that Box VII residues are essential for initiation. Characterization of R281A revealed that its inactivity in initiation is from its inability to properly assemble the prepriming complex. We conclude that strand
opening of oriC is insufficient to provide a structure to which DnaB can stably interact.
Impacts
Chromosomal DNA replication is an essential process that is controlled at the level of initiation of DNA replication. Recent discoveries have revealed surprising similarities in structures and functions between prokaryotic replication proteins and their eukaryotic counterparts. Thus, all organisms share common features in the enzymology of DNA replication. This project investigates the molecular mechanisms of replication proteins during the initiation of DNA replication, and the biochemistry involved in the regulation of this process. Using E. coli as a model organism, important insights into how this process occurs and is regulated in higher organisms have and will be obtained. This information may be important to medicine and agriculture.
Publications
- Simmons, L. A., Breier, A. M., Cozzarelli, N. R., Kaguni, J. M. 2004. Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability. Mol. Microbiol. 51: 349-358.
- Kaguni, J. M. 2004. Chromosomal replication: Initiation at the bacterial origin. Encyclopedia of Biological Chemistry, in press.
- Felczak, M., Kaguni, J. M. 2004. The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin. J. Biol. Chem. 279:51156-51162.
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Progress 01/01/03 to 12/31/03
Outputs
The E. coli replication origin (oriC) is the site at which the enzymatic machinery to duplicate the bacterial genome is assembled. A key component is DnaB protein, the replicative helicase that drives bidirectional replication fork movement. At oriC, DnaA functions in helicase loading; DnaC is also involved because it must be in a complex with DnaB for delivery of the helicase. We showed previously that only two helicase molecules are loaded at oriC, one for each replication fork. As DnaA induces a local unwinding of oriC to load DnaB, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In the last funding period, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. Thus, the availability of single-stranded DNA is not a limiting factor. Other results support a model in which the site of entry for DnaB
is altered so that it cannot be reused. We also showed that 2-4 DnaA monomers are bound at a specific site to load DnaB protein. In E. coli, DnaA protein regulates chromosomal DNA replication by controlling the frequency of initiation from oriC. A mutant form, DnaAcos carrying four amino acid substitutions, is apparently defective in sensing regulatory signals because it induces hyperactive initiation. Recently, we showed that one amino acid substitution (A184V) immediately adjacent to a Walker A box motif causes a defect in ATP binding, leading tyo more frequent initiations. Thus, a defect in ATP binding results in aberrant control of DNA replication. A second amino acid substitution (Y271H) appears to stabilize the activity of the mutant protein carrying the A184V substitution. The hyperinitiation by the dnaAcos allele encoded in single copy by the bacterial chromosome leads to inviability. In contrast, elevated dnaA expression from a regulated promoter in a multicopy plasmid causes
more frequent initiations from oriC, but viability is maintained. We investigated the basis for this discrepancy, and showedby quantitative methods that induced dnaA expression leads to increased initiations. However, the newly formed replication forks fail to progress to completion. Instead, most halt within 10 map units of oriC. Because forks stop randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart was blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression was lethal. The amount of collapsed forks was substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally-encoded dnaAcos is due to hyperinitiation that overwhelms the repair capacity of the cell.
Impacts
Chromosomal DNA replication is an essential process that is controlled at the level of initiation of DNA replication. Recent discoveries have revealed surprising similarities in structures and functions between prokaryotic replication proteins and their eukaryotic counterparts. Thus, all organisms share common features in the enzymology of DNA replication. This project investigates the molecular mechanisms of replication proteins during the initiation of DNA replication, and the biochemistry involved in the regulation of this process. Using E. coli as a model organism, important insights into how this process occurs and is regulated in higher organisms have and will be obtained. This information may be important to medicine and agriculture.
Publications
- Simmons, L. A., Felczak, M. and Kaguni, J. M. 2003. DnaA protein of E. coli: Oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids. Mol. Microbiol. 49: 849-853.
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Progress 01/01/02 to 12/31/02
Outputs
Initiation of DNA replication from the E. coli chromosomal origin, oriC, occurs in a stepwise manner. DnaA protein first binds to DnaA box motifs within oriC, then recruits DnaB helicase to an unwound region. Once DnaB is bound to the separated DNA strands, it translocates and unwinds the parental duplex. Primers formed by primase are then extended by DNA polymerase III holoenzyme. In the last period, we tested if the amount of single stranded DNA in oriC opened by DnaA was limiting and controlled the number of DnaB hexamers that can bind, and proposed that other mechanisms control DnaB binding. Also, we showed that DnaA interacts specifically with HU protein composed of two alpha subunits. As HU assists in unwinding of oriC, we compared the alpha dimer of HU to the beta dimer and the alpha/beta dimer in unwinding of oriC. The results indicate that all forms of HU were equally active. We also found that the moderate level of unwinding of oriC in the absence of added
HU is due solely to DnaA activity. Because DnaA protein appears to regulate the initiation process, we studied a mutant form, DnaAcos, which induces hyperactive initiation and is apparently defective in regulation. We showed that hyperactive initiation is due to two specific amino acid substitutions. One (A184V) next to the Walker A box is defective in ATP binding. The second (Y271H) stabilizes the activity of the mutant protein carrying the A184V substitution. These results indicate that a defective ATP binding results in aberrant DNA replication control. At oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the nonpermissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to oriC. We showed that
the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Using this assay, specific residues in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur. This work has been submitted for publication. In other work, we studied specific dnaA alleles that reduce initiation frequency and chromosome content per cell at 34 to 39oC and fail to support initiation at both 20 and 44oC. Combined with a dnaX mutation, these dnaA mutations suppress the polymerization and growth defects of the dnaX mutant at 39, but not at 42oC. These mutant alleles, designated Cs,Sx for cold-sensitivity and suppression of dnaX, are altered in the binding affinity for both ATP and the DnaA box sequence. Suppression of
dnaX by dnaA appears to be due to reduced initiation activity because the Cs,Sx mutants have the common property of reducing initiation frequency. This work has been submitted for publication.
Impacts
Chromosomal DNA replication is an essential process that occurs by similar biochemical mechanisms in all free-living organisms . This project investigates the molecular mechanisms of initiation of DNA replication and its regulation. The use of E. coli as a model system is important because it provides insight into how DNA replication may occur in other organisms, and how this biochemical pathway is regulated. This work is relevant to the fields of medicine and agriculture.
Publications
- Carr, K.M., Kaguni, J.M. 2002. E. coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin. J. Biol. Chem. 277: 39815-39822.
- Simmons, L.A., Kaguni, J.M. 2003. The dnaAcos allele of Escherichia coli: hyperactive initiation is caused by substitution of A184V and Y271H, resulting in defective ATP binding and aberrant DNA replication control. Mol. Microbiol. 47: 755-765.
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Progress 01/01/01 to 12/31/01
Outputs
DNA replication from the E. coli chromosomal origin, oriC, occurs through an ordered series of events. DnaA protein bound to DnaA box sequences within oriC initiates this biochemical pathway, followed by unwinding of an AT-rich region near the left border. An intermediate (the prepriming complex) then forms requiring the delivery of DnaB helicase of it bound to DnaC protein. Moving on the DNA to unwind the parental duplex, DnaB transiently interacts with primase, resulting in primers that are then extended by the dimeric DNA polymerase III holoenzyme to copy each parental DNA strand. We recently determined that the stoichiometries of DnaA and DnaB bound to oriC are 10 DnaA monomers and 2 DnaB hexamers. That only two DnaB hexamers are bound, one for each replication fork, suggests a mechanism to restrict the loading of additional DnaB hexamers. One model is that the single-stranded DNA in the unwound region of oriC is only sufficient for the binding of two DnaB
hexamers. To test this model, we used a replication system with a single-stranded DNA template carrying a DnaA box sequence in a hairpin structure. On this DNA, only one DnaB hexamer is bound (manuscript in preparation), indicating that the availability of single-stranded DNA does not limit the number of DnaB hexamers that can bind at oriC. HU protein is composed of two subunits. During log phase growth when cells actively support DNA replication, the homodimer of two alpha subunits is present. On transition from log phase to stationary phase growth, the beta subunit is preferentially expressed. The composition of HU changes from that composed of the alpha dimer to the alpha-beta heterodimer to the beta dimer. By DnaA affinity chromatography, we found that the alpha dimer of HU bound to DnaA specifically. We confirmed this interaction in a solid phase binding assay, showing that the alpha homodimer and the alpha-beta heterodimer bound specifically to DnaA protein. The HU beta dimer
bound poorly if at all to DnaA. HU is involved in initiation at oriC by assisting at the step of unwinding. The ratio of HU needed at this step is a few molecules per oriC DNA. These observations suggest that the alpha dimer of HU specifically interacts with DnaA in the recruitment of HU to oriC. Under the model that DnaA protein regulates the initiation process, we developed a genetic method to isolate dnaA alleles that are hyperactive in DNA replication. Seven alleles were obtained and shown biochemically to promote unscheduled initiations from oriC. These mutations map to regions of DnaA protein known or believed to interact with itself or to DnaB, so their hyperactive phenotypes are presumed to relate to a perturbation of these specific functions. The mutant proteins will be studied at the biochemical level to understand how DnaA protein controls the initiation process. Other experiments have focussed on the identification of amino acids of DnaA protein that interact with DnaB. We
have isolated a collection of mutations in a region of DnaA thought to be involved in binding to DnaB. The mutant proteins are inactive in DNA replication using an in vivo assay. These will also be studied biochemically.
Impacts
Chromosomal DNA replication is an essential process that is controlled at the level of initiation of DNA replication. Surprisingly, recent discoveries in the field of DNA replication reveal similarities in structures and functions between prokaryotic replication proteins and their eukaryotic counterparts. As a consequence, all organisms share many common features in the mechanisms of DNA replication. This project investigates the molecular mechanism of replication proteins that act during the initiation of DNA replication, and how they contribute to the regulation of this process. A biochemical understanding of initiation of DNA replication in E. coli as a model organism is important because it provides insight into how DNA replication occurs and is regulated in higher organisms. This information may be important to medicine and agriculture.
Publications
- Ludlam, A.V., McNatt, M. and Kaguni, J.M. 2001. Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase. J. Biol. Chem. 276:27345 27353.
- Carr, K.M. and Kaguni, J.M. 2001. Stoichiometry of DnaA and DnaB proteins in initiation at the E. coli chromosomal origin. J. Biol. Chem. 276:44919-44925.
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Progress 01/01/00 to 12/31/00
Outputs
Identification of an N-terminal domain of DnaC that binds to DnaB. The E. coli DnaB-DnaC complex and not DnaB alone is the active assembly that functions in the entry of DnaB helicase at the bacterial chromosomal origin. This property assigns DnaC the role of ferrying DnaB protein to this chromosomal locus. However, once DnaB is delivered to the chromosomal origin, DnaC must be released from DnaB to unmask the helicase activity of DnaB. A genetic selection method was used to identify the domain of DnaC that interacts with DnaB. The method is based on the premise that the lethal effect observed when dnaC expression is elevated is due to the binding of DnaC to DnaB to inhibit replication fork movement. Mutants that remain viable when dnaC expression is elevated should be defective in this interaction. Missense mutations were obtained that map to a region near the N-terminus, suggesting that the corresponding natural amino acids normally interact directly with DnaB.
Corresponding mutant proteins were shown to be unable to bind to DnaB and are inactive in DNA replication as a consequence. As further proof of their functional defect, these alleles were also impaired in complementing the temperature-sensitive phenotype of a dnaC28 mutant. In contrast, these mutant proteins were active in ATP binding as indicated by UV-crosslinking to radiolabelled ATP. Amino acids near the N-terminus of DnaC (arginine-10, leucine-11, leucine-29, tryptophan-32, serine-41 and leucine-44) that reside in regions of predicted secondary structure (PHD method) apparently bind directly to DnaB in forming the DnaB-DnaC complex. Other mutations that map to regions bearing Walker A and B boxes are apparently defective in ATP binding, a required step in DnaB-DnaC complex formation. Lastly, DNA sequencing showed that the sixth codon from the N-terminus encodes aspartate, in agreement with N-terminal amino acid sequence data. This resolves the discrepancy between the predicted
amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama et al (1987) J. Biol. Chem. 262, 10475-10480). Stoichiometries of DnaA, and DnaB in the prepriming complex. We previously reported on experiments that determined the stoichiometry of several replication proteins in an intermediate of the replication process, termed the prepriming complex. This intermediate is formed on a single stranded DNA that is duplicated via a process that is very similar to that which occurs at the E. coli chromosomal origin. Comparable experiments were performed to determine the stoichiometry of replication proteins in the prepriming complex that assembles at the E. coli chromosomal origin. The stoichiometries of DnaA and DnaB protein were determined under several experimental conditions. The important observation is that the number of DnaB molecules in the prepriming complex is restricted, suggesting a mechanism to control this event.
Impacts
Chromosomal DNA replication is essential in all free-living organisms. This highly regulated process is controlled at the level of initiation of DNA replication. This project examines the functions of different replication proteins during the stage of initiation of DNA replication. A biochemical understanding of the process of initiation of DNA replication is significant because it provides insight into how DNA replication occurs in higher organisms. This information may be important to medicine and agriculture.
Publications
- Ludlam, A. V., McNatt, M. Carr, K. Kaguni, J.M. 2001. Essential Amino Acids of E. coli DnaC Protein Involved in Interaction with DnaB Helicase. Mol. Micro. submitted
- Ludlam, A. V. McNatt, M. Kaguni, J.M. 2001. An N-terminal domain of E. coli DnaC interacts with DnaB helicase. Mol. Micro. submitted
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Progress 01/01/99 to 12/31/99
Outputs
Identification of amino acids of DnaC that are important for interaction with DnaB--The essential role of DnaC protein in bacterial DNA replication is well established but its biochemical functions are poorly defined and limited to the following observations. DnaC binds to DnaB to form the DnaB6-DnaC6 complex. The binding of ATP to each DnaC monomer is required. On entry of DnaB at the E. coli chromosomal origin,oriC, ATP hydrolysis releases DnaC, a step that is essential to unmask the helicase activity of DnaB. An optimal ratio of DnaC to DnaB is critical for DNA synthesis; excess DnaC is inhibitory apparently because this inhibits the helicase activity of DnaB. The limited information on the role of DnaC in DNA replication prompted studies to obtain novel dnaC alleles to learn more about DnaC function. Relying on the lethality caused by elevated dnaC expression, we selected novel dnaC mutations that were no longer lethal when dnaC expression was elevated to the
normally toxic level. Several classes of mutations are expected. As ATP binding by DnaC is required to form the DnaB-DnaC complex, one class is those that fail to bind ATP. Those that bind ATP but fail to hydrolyze it are lethal and will not be obtained. A second class is those have a higher ATPase activity to more effectively release DnaC from DnaB. The third encodes amino acid substitutions at residues that normally act in binding to DnaB. These should be viable because the mutant DnaC protein is unable to bind to DnaB to inhibit its activity. Several dnaC alleles have been isolated and characterized. DNA sequence analysis suggests that some alleles encode proteins that are defective in ATP binding because they affect amino acid residues in a motif proposed to be involved in ATP binding. Other missense alleles may be defective in binding to DnaB. Biochemical experiments are in progress to characterize the mutant proteins. These studies have the potential to reveal novel functions of
DnaC protein in the replication process, including the identification of a functional domain that interacts with DnaB. Stoichiometries of DnaA, DnaB and DnaC in a replicative intermediate--Experiments were done to determine the stoichiometry of DnaA, DnaB, and DnaC in an intermediate of the replication process termed the prepriming complex. When isolated, this intermediate supports DNA replication on addition of replication proteins that act subsequently. We wanted to know if DnaA promoted the binding of one or many DnaB hexamers from the DnaB-DnaC complex. In the intermediate formed on a single-stranded with the DnaA box in a hairpin structure, quantitative immunoblot analysis showed that 6-7 monomers of DnaA are bound per ssDNA. DnaB helicase was present at 1 copy. The finding of only a single DnaB hexamer bound per ssDNA suggests that a mechanism regulates the number of DnaB hexamers that can bind. If so, this may be an important regulatory step in the initiation process. We are
confirming these stoichiometries and will shortly begin the stoichiometric determination of these proteins in prepriming complexes formed on an oriC plasmid.
Impacts
Chromosomal DNA replication is an essential cellular process that is strictly coupled to cell growth, and is controlled at the stage of initiation of DNA replication . This work investigates the roles of different replication proteins in the initiation process. Insight from these studies may be of medical and agricultural importance as they relate to the initiation process in higher organisms.
Publications
- Simmons, L.A. and Kaguni, J.M. 1999. Selection method for isolation of dnaA mutants that are hyperactive in the initiation of DNA replication. Molecular Mechanisms in DNA Replication and Recombination. p. 78.
- Vicente, M. and Kaguni, J.M. 1999. Proteins that interact with E. coli DnaA protein and modulation of chromosomal DNA replication. Molecular Mechanisms in DNA Replication and Recombination. p. 79.
- Carr, K. and Kaguni, J.M. 1999. Interaction of the initiator protein, DnaA, with the DnaB helicase in formation of prepriming complexes at a prokaryotic origin of replication. Molecular Mechanisms in DNA Replication and Recombination. p. 65.
- Ludlam, A.V., Basa, J.D. and Kaguni, J.M. 1999. Amino acid residues of E. coli DnaC protein that interact with DnaB helicase. Molecular Mechanisms in DNA Replication and Recombination. p. 74.
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Progress 01/01/98 to 12/31/98
Outputs
The duplication of chromosomes in all free-living organisms is tightly coordinated with cell division and is regulated at the stage of initiation of the process. One major question is to determine how this process is regulated. This event starts by the step-wise assembly of a nucleoprotein complex termed the replisome at specific sites in the genome called origins of replication. In replisome assembly, an intermediate called the prepriming complex is formed that functions in the entry of the replicative helicase. The helicase then acts at the newly formed replication forks to unwind the parental duplex in advance of DNA replication. We have established a model in vitro system composed of highly purified proteins and a supercoiled plasmid template carrying a chromosomal origin that has revealed the individual events at the stage of initiation. In this system, Escherichia coli DnaA protein acts as the replisome organizer by its binding to specific DNA sequences in the
replication origin to promote the formation of the prepriming complex. An important event involves the entry of DnaB, the replicative helicase. We studied how this occurs, and identified a domain near the N-terminus that participates in the entry of DnaB, and is highly conserved among 25 dnaA homologs. Mutant proteins lacking this N terminal region are specifically defective in the proper recruitment of DnaB helicase into the prepriming complex, but are active in other functions. We propose that a specific nucleoprotein structure is formed by DnaA protein that is important for the entry of DnaB at the chromosomal origin. The E. coli chromosomal origin bears several bindings sites for FIS, a protein first discovered by its role in site-specific DNA recombination. The strongest FIS binding site is closely flanked by two sites for DnaA protein that are required for the initiation of chromosomal replication. Due to their closeness, we considered the possibility that FIS affects the
initiation process by modulating DnaA protein binding. However, our studies show that the binding of FIS does not block the binding of DnaA protein nor does FIS inhibit in vitro DNA replication from the chromosomal origin. Inhibition, observed only at high levels of FIS, is by sequestering the negative superhelicity of the plasmid DNA that is essential for initiation. These observations suggest that if FIS in the cell acts to regulate initiation from the chromosomal origin, it is indirect. As a monomer, DnaA protein binds ATP with high affinity and is active in DNA replication. As a self-aggregate, it is inactive in ATP binding and its replication activity is greatly diminished. Because the physical state of DnaA profoundly influences its activity in DNA replication, we studied factors that stabilized the monomer form. We found that ATP or ADP but not AMP or cAMP stabilized monomeric DnaA. Chaperones DnaK or GroEL/ES also prevented aggregation that otherwise occurred fairly rapidly
(50% aggregation in 30 sec.). Factors in the cell that affect the physical form of DnaA protein may have a regulatory affect on the initiation process.
Impacts
(N/A)
Publications
- Banecki, B., Kaguni, J.M., Marszalek, J. 1998. Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli. Biochim. Biophys. Acta. 1442: 39-48.
- Sutton, M. D., Carr, K.M., Vicente, M., Kaguni, J.M. 1998. Escherichia coli DnaA protein: The N-terminal domain and loading of DnaB helicase at the E. coli chromosomal origin. J. Biol. Chem. 273: 24355-34262.
- Margulies, C., Kaguni, J.M. 1998. The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin. Nucleic Acids Res. 26: 5170-5175.
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Progress 01/01/97 to 12/31/97
Outputs
DnaA protein of E. coli is the initiator of chromosomal DNA replication. Because DNA replication is regulated during the initiation process, this protein has been the focus of study with long range objectives of understanding biochemically the initiation of chromosomal replication, and its regulation. In the previous reporting period, we isolated and characterized novel mutant forms of DnaA protein in order to correlate structural domains of DnaA protein to its various functions. This study revealed four functional domains of DnaA protein involved in DNA binding, ATP binding, in replication of pSC101 (an E. coli plasmid that relies on host replication proteins for its propagation), and in forming the proper nucleoprotein complex at the replication origin for the binding of DnaB, the replicative helicase responsible for progression of the replication forks.
Impacts
(N/A)
Publications
- Kaguni, J. M. 1997. Escherichia coli DnaA Protein: The replication initiator. Mol. Cells 7: 145-157
- Sutton, M. D., and Kaguni, J.M. 1997. Novel alleles of the E. coli dnaA gene. J. Mol. Biol. 271: 693-703
- Sutton, M. D., and Kaguni, J.M. 1997. Threonine 435 of E. coli DnaA protein confers sequence-specific DNA binding activity. J. Biol. Chem. 272: 23017-23024
- Sutton, M. D., and Kaguni, J.M. 1997. The E. coli dnaA gene: four functional domains. J. Mol. Biol. 274: 546-561
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Progress 01/01/96 to 12/30/96
Outputs
Over 20 different genes are required for replication of the Escherichia coli chromosome. Among these, the dnaA gene is unique in that it is solely required to initiate this process. By focussing on the role of the dnaA gene product, the long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. One major approach is to isolate and characterize novel mutant forms of DnaA protein. The second is to correlate structural domains of DnaA protein to its various functions. From the study of a collection of dnaA mutants by genetic and biochemical methods, four functional domains compose DnaA protein. Whereas we have not correlated a biochemical function to one domain, the remaining three are involved in DNA binding, ATP binding, and in replication of pSC101. The latter is a plasmid of E. coli that requires DnaA protein for its replication. Other studies indicate that DnaA protein binds to sites in the
E. coli chromosomal origin in an ordered and sequential manner. Binding of DnaA protein to the last site, DnaA box R3, is critical in that it correlates with replication activity. Finally, we have characterized biochemically a mutant form of DnaA protein with a substitution at alanine 184 with valine. This mutant protein is defective in ATP binding, and requires activation by DnaK and GrpE heat shock proteins to observe ATP binding and replication activity.
Impacts
(N/A)
Publications
- MARGULIES, C., KAGUNI, J.M. 1996. Ordered and sequential binding of DnaA proteinto oriC, the chromosomal origin of E. coli. J. Biol. Chem. 271:17035-17040.
- MARSZALEK, J. ZHANG, W., HUPP, T.R., MARGULIES, C. M., KAGUNI, J.M. 1996. Domainsof DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin. J. Biol. Chem. 271:18535-18542.
- BURTON, Z. F., KAGUNI, J.M. 1997. Experiments in Molecular Biology: Biochemical Applications (text for an undergraduate laboratory course) Academic Press, San Diego, CA (in press).
- SUTTON, M., KAGUNI, J.M. 1997. Novel alleles of the E. coli dnaA gene. Manuscript in preparation.
- SUTTON, M., KAGUNI, J.M. 1997. The E. coli dnaA gene: four functional domains. Manuscript in preparation.
- SUTTON, M., KAGUNI, J.M. 1997. Threonine 435 of E. coli DnaA protein confers sequence-specific DNA binding activity. Manuscript in preparation.
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Progress 01/01/95 to 12/30/95
Outputs
The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. The approaches taken are i) to isolate and characterize novel mutant forms of DnaA protein, and ii) to correlate structural domains of DnaA protein to its various functions. Escherichia coli DnaA protein binds to the chromosomal origin to initiate chromosomal DNA replication. Comparative analysis of 15 DnaA protein homologs has led to the suggestion that conserved domains are important for its structure or activity. In the past 2 years, we have studied genetically and biochemically mutants we obtained by a novel genetic method. Mutations that substitute conserved residues reduce or abolish DNA replication activity (manuscript in preparation). Further study has identified domains involved in DNA binding, ATP binding, and in replication of pSC101 (manuscript in preparation). The latter is a plasmid of E. coli that requires DnaA
protein for its replication. These studies are complemented by an approach that utilizes monoclonal antibodies to DnaA protein (submitted). One antibody blocks the interaction between DnaA protein and DnaB helicase. The epitope recognized by the antibody has been determined. This study suggests a region of DnaA protein that interacts specifically with DnaB protein. This observation is important in light of our finding that DnaA protein binds in an ordered manner to the chromosomal origin (submitted).
Impacts
(N/A)
Publications
- SUTTON, M., KAGUNI, J.M. 1995. Novel mutants of the E. coli dnaA gene are defective in pSC101 replication. J. Bacteriol. 177, 6657-6665.
- CARR, K.M., KAGUNI, J.M. 1996. The A184V missense mutation of the dnaA5and dnaA46 alleles confers a defect in ATP binding and thermolability in initiation of E. coli DNA replication. Submitted.
- MARSZALEK, J., ZHANG, W., HUPP, T.R., MARGULIES, C.M., KAGUNI, J.M. 1996. Characterization of monoclonal antibodies which interact with DnaA protein. Submitted.
- MARGULIES, C., KAGUNI, J.M. 1996. DnaA protein binds in an ordered and sequential manner to oriC, the chromosomal origin of E. coli. Submitted.
- BURTON, Z.F., KAGUNI, J.M. 1995. Experimental Approach to Molecular Biochemistry. (Text for an undergraduate laboratory course) to be published by Academic Press.
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Progress 01/01/94 to 12/30/94
Outputs
The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. The approaches taken are i) to isolate and characterize novel mutant forms of DnaA protein, and ii) to correlate structural domains of DnaA protein to its various functions. Escherichia coli DnaA protein binds to the chromosomal origin to initiate chromosomal DNA replication. Amino acid sequence conservation of regions of DnaA protein, deduced by comparative analysis of homologs from other bacteria, suggests domains involved in its various biochemical activities. We have characterized a collection of mutants of the dnaA gene. These mutants were obtained by a novel genetic method. The expectation of this approach was to identify residues essential for activity. Domains of DnaA protein involved in DNA binding, and ATP binding have been identified. In conjunction, we have mapped epitopes recognized by a collection of monoclonal
antibodies to DnaA protein. One monoclonal antibody that interferes with the interaction between DnaA protein and DnaB helicase implicates a region of DnaA protein involved in this interaction. Finally, biochemical characterization of DnaA protein indicates that it binds in an ordered manner to the chromosomal origin.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. The approaches taken are i) to isolate and characterize novel mutant forms of DnaA protein, and ii) to correlate structural domains of DnaA protein to its various functions. Escherichia coli DnaA protein of 467 amino acid residues binds to the chromosomal origin to promote DNA replication. Comparison of DnaA protein homologs from other bacteria reveals a striking degree of sequence conservation. Conserved residues may be important for structure and/or function in the initiation of DNA synthesis. By a novel genetic method, we have obtained a large collection of mutants and are characterizing them in comparison to the wild type protein and other mutant forms that we have studied. This approach is expected to identify residues essential for activity. In conjunction, we have isolated a collection of monoclonal antibodies to DnaA
protein. The epitopes recognized have been determined. Functional studies with these antibodies have also been performed. One monoclonal antibody has been of particular interest. We showed it to interfere with the interaction between DnaA protein and DnaB helicase. This replication protein acts subsequently in the replication process to unwind the parental duplex DNA in advance of movement of the replication fork. From this study, one role of DnaA protein is to promote the binding of DnaB helicase.
Impacts
(N/A)
Publications
- ZEHNER, M.D. 1993. What the consumer is saying about flats, pack sizes and container shapes for annual flowering plants. Ag. Econ. Staff Report 93-52. pp.
- ZEHNER, M.D. 1993. Listing of municipally and community sponsored retail farmers' markets in Michigan. Ag. Econ. Staff Report 93:49. 19 pp.
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Progress 01/01/92 to 12/30/92
Outputs
The long range objectives of this research are to understand biochemically the initiation of chromosomal replication, and its regulation. Two approaches taken to address this problem are i) to characterize mutant forms of DnaA protein, and ii) to correlate structural domains of DnaA protein to its various functions. The above aims depend on sufficient amounts mutant or altered forms of DnaA protein. We have recently constructed expression vectors in which wild type or mutant dnaA genes have been placed under T7 promoter control. These strains have facilitated the purification of wild type DnaA protein and novel mutant forms. As a prelude to the study of novel dnaA mutants, we determined the biochemical defect of a dominant-lethal mutant of dnaB. The involvement of DnaK protein in replication described in the previous progress report, and observations of others that expression of dnaK in Caulobacter crescentus occurs at a specific time in the cell cycle concomitant with
DNA synthesis prompted an examination of the temporal expression of dnaK in E. coli. By use of a synchrony method, the rate of synthesis of DnaK protein did not change dramatically relative to the cell cycle (manuscript in preparation). Genetic and physiological evidence indicate that RNA polymerase participates in some aspect of the initiation process.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
DNA replication in Escherichia coli, as in most organisms, is regulated at the level of initiation of DNA replication and is tightly coordinated to cell growth. The objectives of this work are to understand the biochemical mechanism of initiation of replication, the role of dnaA protein in this process, and its coordination to the bacterial growth cycle. The experimental approaches are 1) to characterize mutant forms of dnaA protein in comparison with dnaA+ protein, and 2) to use monoclonal antibodies against dnaA protein in the study of dnaA protein function. Progress in the last granting period includes purification of a factor and its identification as grpE protein. In concert with dnaK protein, it is required for replication activity of gene products encoded by the dnaA5 and dnaA46 alleles. Previous experiments indicated that mutant forms of dnaA protein are thermolabile in DNA replication. We have determined that the interaction of dnaA5 and dnaA46 protein with
dnaK and grpE protein represents the thermolabile deficiency associated with these mutant forms. Third, a collection of monoclonal antibodies against dnaA protein have been isolated. dnaA+ protein, a sequence-specific DNA binding protein, also binds ATP with high affinity. About half of these antibodies inhibit ATP binding. Only four inhibit DNA replication. None appear to inhibit the ability of dnaA protein to bind to restriction fragments containing the chromosomal origin sequence.
Impacts
(N/A)
Publications
- HWANG, D.S., and KAGUNI, J.M. (1991). dnaK protein stimulates a mutant form of dnaA protein in Escherichia coli DNA replication. J. Biol. Chem. 266. 7537-7555.
- MARSZALEK, J., and KAGUNI, J.M. (1991). Defective replication activity of a dominant-lethal dnaB gene product from Escherichia coli. J. Biol. Chem. accepted for publication.
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Progress 01/01/90 to 12/30/90
Outputs
A collection of monoclonal antibodies against Escherichia coli dnaA protein, a site-specific DNA binding protein which plays a key role in initiation of chromosomal replication, have been purified and characterized with regard to IgG subclass and the epitope class recognized by each. Experiments have been performed to determine activities of dnaA+ protein which these antibodies inhibit. dnaA+ protein binds ATP with high affinity (K(subscript D ) 0-03 (mu)M). This complex is active in initiation of DNA replication by unwinding the chromosomal origin. Four of these antibodies inhibit DNA replication apparently as a consequence of inhibition of ATP binding. Five others inhibit DNA replication presumably as a result of inhibition of unwinding of the chromosomal origin by dnaA protein. 20-40 monomers of dnaA protein bind cooperatively to oriC. None appear to inhibit the ability of dnaA protein to bind to restriction fragments containing the chromosomal origin sequence,
oriC, as measured by nitrocellulose filter binding assays. This assay does not distinguish between the binding of a single monomer or many molecules of dnaA protein. It is possible that some of these antibodies interfere with DNA replication by interfering with the cooperative nature of dnaA protein binding to oriC. This possibility will be examined by performing DNase I or hydroxyl radical footprinting.
Impacts
(N/A)
Publications
- HWANG, D.S., CARR, K.M., and KAGUNI, J.M. 1990. dnaK protein interacts with dnaA protein in Escherichia coli DNA replication. J. Biol. Chem., accepted with revisions.
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