Progress 01/01/15 to 12/31/19
Outputs
Target Audience:The target audience of the work is other research investigators who work in the areas of DNA replication, DNA repair, and in related areas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Drs. Chodavarapu and Felczak are postdoctoral fellows who conducted many of the experiments in the peer-reviewed publications of this report. The work of these research articles is part of their training and professional development. How have the results been disseminated to communities of interest? The peer-reviewed research articles and review articles have been published in open access journals. and are available to the scientific community and the public. All publications from the Kaguni lab may also be obtained upon request by email. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Mechanistic studies DNA replication is essential in all free-living organisms, and is strictly coordinated with cell growth. Our ability to manipulate this process to promote or block cell growth, as in the case of infection or cancer, requires a mechanistic understanding that includes the function of each individual replication protein. The focus of the Kaguni 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. One mechanism of regulation is through Hda and the dimeric β clamp, which stimulate the hydrolysis of ATP bound to DnaA. The resulting DnaA-ADP is not active in replication initiation. In collaborative work with two other labs, we reported on the crystal and solution structure of the Hda-β clamp complex that contains two pairs of Hda dimers sandwiched between two β clamp rings. Among the three interacting surfaces between Hda and the β clamp, and between Hda protomers, the interface between Hda monomers occludes a critical arginine residue that is involved in the hydrolysis of ATP bound to DnaA. Hence, Hda in the octameric complex is unable to regulate the activity of DnaA. This study indicates that the octameric complex must disassemble to permit the arginine residue of Hda to interact with DnaA. At a DNA replication origin, helicase loading often requires the dynamic interactions between the DNA helicase, a ring-shaped enzyme, and a companion protein. In Escherichia coli, the DNA helicase is DnaB and DnaC is its loading partner. In a recent studies, we addressed the importance of DnaB-DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring naturally opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring. Evidence indicates that DnaC alters the helical hairpins in the N-terminal domain of DnaB to occlude this region from interacting with primase. Other observations suggest that DnaC and primase have opposing effects on each other. Apparently, the binding of DnaC or primase to the respective domains of DnaB transduces a conformational change to the other domain to interfere with binding of the second protein. On the basis of the dynamic interactions of DnaC and primase with DnaB, we suggest the novel idea that DnaC controls the access of DnaB to primase. Separate work focused on the binding of DnaC to single-stranded DNA, which was speculated to be required for DnaC to function in DNA replication. Genetic and biochemical characterization of mutant proteins designed to be defective in DNA binding revealed an unexpected increase in affinity for DNA. Apparently, the gain-of-function mutations cause DnaC to become defective in DNA replication. We suggest that the impaired ability to dissociate from the single-stranded DNA causes DnaC to remain bound to DnaB, which blocks its activation as a DNA helicase. Development of novel antibiotics In free-living organisms, the enzymes that function at the respective stages of DNA replication differ in amino acid sequence, and their three-dimensional structures. Among bacteria, protein homologues are highly conserved. Hence, compounds that target this essential pathway should be specific to bacteria. A long-term aim is to identify compounds that inhibit DNA replication in bacteria with the goal of developing them into novel antibiotics. Much attention has been given to the transfer of multi-drug resistant bacteria from food animals to humans, and the threat of such bacteria to human health. Acknowledging the danger, the World Health Organization in 2017 issued a tiered list of multidrug-resistant pathogens. However, infectious diseases also imperil food animals. Using the example of bovine respiratory disease, it is the most common and costly disease in the U.S. beef cattle industry. The emergence of pathogens resistant to drugs used to treat the respiratory disease has increased in feedlot cattle. Thus, new antibiotics that are effective against multi-drug resistant bacterial are critical to the food animal industry. Recently, Kaguni's lab established and optimized a high-throughput assay system that requires almost all of the same proteins required for duplication of the E. coli chromosome. Using this assay, we identified compounds that specifically inhibit DNA replication. These compounds will be studied further.
Publications
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience is other research investigators in the field of biochemistry and molecular biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Drs. Chodavarapu and Felzcak performed experiments in the published reports and manuscripts under review as part of their training and professional development. As part of their scientific training, they also helped to analyze the data, and write the manuscripts with Dr. Kaguni. How have the results been disseminated to communities of interest? The publications and manuscripts under review are in "open access" journals that are accessible by other research investigators. The publication reported in this funding period and past publications are available on request by email, or by subscription. What do you plan to do during the next reporting period to accomplish the goals?One major objective is to determine the structure of a complex containing DnaA, DnaB and DnaC that is formed in the process of replication initiation. Biochemical evidence indicates that the complex contains a DnaA monomer assembled on a DnaA box-containing hairpin structure in a single-stranded DNA (ssDNA) fragment (390 nucleotides). A single DnaB6-DnaC6complex is thought to be positioned at the base of the hairpin. By hydrogen/deuterium exchange analysis of the complex, we will identify the surfaces of these proteins that interact. We will also determine its cryo-electron microscopic structure by collaborating with another lab that specializes in this method. To obtain 3D reconstruction of 2D class averages of the images collected, we will integrate structural homology models of E. coli DnaA and the E. coli DnaB-DnaC complex. These homology models have been built using X-ray structures of Geobacillus stearothermophilus DnaB and the AAA+ domain of Geobacillus kaustophilus DnaA as templates. An understanding of the relative positions and orientations of DnaA, DnaB and DnaC in the complex is essential to understand the mechanism of helicase loading.?
Impacts
What was accomplished under these goals?
DnaB is the replicative DNA helicase inEscherichia colithat is responsible for unwinding the parental DNA, a process that is required for the DNA to be copied during DNA replication. A partner protein named DnaC regulates the function of DnaB, but the mechanism of regulation is poorly understood. In earlier work, we found that the binding of DnaC to DnaB causes a dramatic conformational change of the N-terminal domain of DnaB. This domain is recognized and bound by primase; formation of a complex of primase with DnaB is essential for primer formation by primase. These findings suggest that the conformational change of the N-terminal domain of DnaB inhibits the binding of primase. We tested this model, and obtained evidence to support it using the independent methods of biosensor analyses, enzyme-linked immunosorbent assays, and hydrogen/deuterium exchange analyses. Other experiments showed that a specific amino acid in the N-terminal domain of DnaB likely interacts directly with primase. Substitution of this residue with alanine specifically impairs the interaction between the helicase and primase. In contrast, other substitutions alter the conformation of a sub-domain named the helical hairpin and/or its pairing with the counterpart subdomain in the companion DnaB protomer to interfere with the binding of DnaB to ssDNA and to primase. Hence, primer formation and subsequent DNA replication are inhibited. On the basis of the dynamic interactions of DnaC and primase with DnaB, we suggest a model in which DnaC controls the access of DnaB to primase. This study offers important new insights on elegant control mechanisms during replication initiation. The fundamental strategies to duplicate chromosomes are similar in all free-living organisms, but the respective enzymes that are functional counterparts in DNA replication differ in amino acid sequence, their three-dimensional structures, and generally utilize different enzymatic mechanisms. Hence, the replication proteins that are highly conserved among bacterial species are attractive targets to develop novel antibiotics as the compounds are unlikely to demonstrate off-target effects.Escherichia colihas been developed as a model system to study DNA replication, serving as a benchmark for comparison. In work to identify novel compounds that inhibit bacterial DNA replication so that they may be developed into antibiotics that target this essential pathway, we wrote a summary of the functions of individualE. coliproteins, and the compounds that inhibit them. TheEscherichia colidnaNgene encodes thebclamp that, when bound to theE. colichromosome, helps to recruit partner proteins to the DNA. Using a genetic selection, sixdnaNalleles were obtained that confer resistance to hydroxyurea when expressed at endogenous levels. To understand the mechanistic basis for the phenotype of hydroxyurea resistance, the mutant bearing an E202K substitution (dnaNE202K) was chosen for further study. We found that thednaNE202Kstrain expresses an elevated level of the DnaA-regulated class 1a ribonucleotide reductase encoded bynrdAB, explaining why it is resistant to HU. ThednaNE202Kmutant also expresses altered levels of other DnaA-regulated genes, and underinitiates DNA replication. Becausethebclamp complexed to Hda interacts with ATP-bound DnaA (DnaA-ATP) to stimulate the hydrolysis of ATP, resulting in DnaA-ADP that is not active in replication initiation, these observationssuggest thatbE202Ktogether with Hda leads to a reduced level of DnaA-ATP relative to DnaA-ADP. In contrast with these in vivo results, thebE202Kclamp was comparable in vitro with the wild type clamp in stimulating the hydrolysis of ATP bound to DnaA. Biosensor experiments revealed thatbE202Kbinds with comparable affinity as the wild type clamp to Hda and thedsubunit of the DnaX clamp loader, but displays a greater affinity for DNA Pol III. A manuscript discussing these results is under revision.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Felczak MM, Chodavarapu S, Kaguni JM. (2017) DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase. J Biol Chem. 292: 20871-20882. doi: 10.1074/jbc.M117.807644. Epub 2017 Oct 25, PMID:29070678
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Kaguni, J.M. (2018) The macromolecular machines that duplicate the Escherichia coli chromosome as targets for drug discovery. Antibiotics 7 doi:10.3390/antibiotics7010023, PMID: 29538288, PMCID: PMC5872134
|
Progress 10/01/16 to 09/30/17
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 Felzcak performed experiments in the published reports and manuscripts under review as part of their training and professional development. As part of their scientific training, they also helped toanalyzethe data, andwrite the manuscripts with Dr. Kaguni. How have the results been disseminated to communities of interest?The publications and manuscripts under review are in "open access" journals that are accessible by other research investigators. The publication reported in this funding period and past publications are available on request by email, or by subscription. What do you plan to do during the next reporting period to accomplish the goals?Our objectives are to determine the structure of a complex containing DnaA, DnaB and DnaC that is formed in the process of replication initiation. As summarized in last year's progress report, this complex has a distinct composition of a DnaA monomer assembled on a DnaA box-containing hairpin structure in a single-stranded DNA (ssDNA) fragment (390 nucleotides). It also contains a single DnaB6-DnaC6 complex, which is loaded onto the DNA by DnaA. To understand the process of assembly of this complex at the molecular level, we will determine the interacting surfaces of these proteins in the complex following the method of hydrogen/deuterium exchange analysis that is already established in our lab. We will also determine its cryo-electron microscopic structure by collaborating with another lab that specializes in this method. Relying on structural homology models of E. coli DnaA and the E. coli DnaB-DnaC complex using X-ray structures of Geobacillus stearothermophilus DnaB and the AAA+ domain of Geobacillus kaustophilus DnaA as templates, we will integrate the cryo-electron microscopy results in combination with 3D image reconstruction of the complex. This work to establish the relative positions and orientations of DnaA, DnaB and DnaC in the complex is essential to understand the mechanism of helicase loading.
Impacts
What was accomplished under these goals?
The regulatory inactivation of DnaA (RIDA) is a major mechanism that regulates the frequency of replication initiation. In RIDA, it is presumed that an Hda monomer interacts with the β clamp (a dimer) bound to DNA to form a complex that directly interacts with ATP-bound DnaA (DnaA-ATP). This interaction stimulates the hydrolysis of ATP, resulting in DnaA-ADP that is not active in replication initiation. A prediction is that the activity of Hda is tightly controlled in a cell cycle-dependent manner to ensure that replication initiation occurs only once and at the proper time of the cell cycle. In collaborations with two other labs, we published a study that describes the crystal structure of an octameric Hda-β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings. Among the three interacting surfaces between Hda and the β clamp, and between Hda protomers, one interface between Hda monomers occludes a critical arginine residue that is involved in the hydrolysis of ATP bound to DnaA. Hence, Hda in this octameric complex, whose functional importance is supported by biochemical experiments and mutational evidence, is unable to regulate the activity of DnaA. This study reports on a novel mechanism that regulates the function of Hda. In addition to the interaction of the β clamp with Hda described above, the βclamp interacts with other proteins to coordinate their activities in DNA replication, DNA repair and damage tolerance. One of these interacting proteins is DNA polymerase III holoenzyme, the cellular replicase that is responsible for copying the bacterial chromosome. Studies performed in collaboration with another lab showed that an elevated level of the β clamp, but not of mutant clamps carrying specific amino acid substitutions, slowed growth by impeding DNA replication. These observations suggest that an elevated level of the β clamp may sequester one or more essential replication proteins away from the fork. These and other results support a model in which Hda outcompetes with DNA Polymerase III for binding to mutant β clamps bound to the bacterial chromosome to reduce the level of DnaA-ATP. A manuscript describing these results is under revision. DnaB is the replicative DNA helicase of Escherichia coli. At the initiation stage of DNA replication, DnaB must be in a complex via its C-terminal domain (CTD) with DnaC for subsequent helicase loading at the replication origin of the E. coli chromosome. During initiation at the replication origin and on the lagging strand DNA template in the formation of Okazaki fragments, the N-terminal domain (NTD) of DnaB is thought to serve as the landing pad for primase. This interaction is required for synthesis of primers needed by DNA polymerase III for DNA replication. At the replication origin, primer formation induces the dissociation of DnaC from DnaB, leading to activation of DnaB as a DNA helicase. Unfortunately, we have a poor understanding of this fundamental process of helicase loading and helicase activation. In the manuscript under review, we describe several important discoveries. First, we showed that DnaC impairs the binding of primase to DnaB, which correlates with an altered conformation of a subdomain named the helical hairpin in the NTD of DnaB. These findings suggest a model that the binding of DnaC to the CTD of DnaB alters the conformation of DnaB's NTD to inhibit the binding of primase. Second, we showed that specific amino acid substitutions in subdomains of this region lead to the inability of DnaB to interact with primase, confirming the model. These observations suggest that DnaC and primase have opposing effects on each other. Apparently, the binding of DnaC or primase bind to respective domains of DnaB transduces a conformational change to the other domain to interfere with binding of the second protein. On the basis of the dynamic interactions of DnaC and primase with DnaB, we suggest a model in which DnaC controls the access of DnaB to primase. This study offers important new insights on elegant control mechanisms during replication initiation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Kim, J.S., Nanfara, M.T., Chodavarapu, S., Jin, K.S., Babu, V.M., Ghazy, M.A., Chung, S., Kaguni, J.M., Sutton, M.D., and Cho, Y. (2017) Dynamic assembly of Hda and the sliding clamp in regulation of replication licensing. Nucleic Acids Res. 45, 3888-3905, doi: 10.1093/nar/gkx081, PMID: 28168278
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Babu, V.M.P., Chodavarapu, S., Nanfara, M.T., Maul, R.W., Kaguni, J.M., and Sutton, M.D., The ? clamp and Hda regulate deoxynucleotide levels via DnaA. manuscript under revision.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Felczak, M.M., Chodavarapu, S., and Kaguni, J.M. DnaC controls the accessibility of DnaB by primase. manuscript under review
|
Progress 10/01/15 to 09/30/16
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?As postdoctoral fellows, Drs. Chodavarapu and Felczak performed the experiments in the published reports as part of their training and professional development. Together with Dr. Kaguni, they also helped write the manuscripts, and analyzed the data as part of their instruction in scientific writing. How have the results been disseminated to communities of interest?The publications in "open access" journals are accessible by other research investigators. For these and other individuals, these publications are also available on request by email, or by subscription. What do you plan to do during the next reporting period to accomplish the goals? Our objectives are to identify the segment(s) within the N-terminal domain of DnaA that interacts with DnaB during the process of helicase loading, and to understand how these and other proteins interact during the initiation of DNA replication. Our experimental approach is to study an intermediate formed in the process of replication initiation named the ABC complex. Formed in the presence of ATP or ATPgS, the ABC complex contains 4-5 DnaB monomers and 4-5 DnaC monomers, which correlates with the well-characterized DnaB6-DnaC6 complex. In unpublished biosensor experiments using a biotinylated single-stranded DNA (ssDNA) fragment (390 nucleotides) that carries the hairpin (62 nucleotides) immobilized on a streptavidin-coated sensor chip, it retained 4.5 SSB molecules at saturation, which conforms with the (SSB)65 mode of binding to the amount of ssDNA, and the appearance of four complexes that differ incrementally in their mobility in gel mobility shift experiments. In biosensor experiments with increasing concentrations of DnaA, 0.93 molecule/DNA bound to the SSB-coated ssDNA at saturation, consistent with a single DnaA monomer at the hairpin. Similar results were obtained by quantitative immunoblotting of the isolated ABC complex. The subsequent addition of the DnaB-DnaC complex showed that it is also able to bind. In contrast, we observed negligible binding of DnaA or of the DnaB-DnaC complex following DnaA with an SSB-bound control ssDNA (340 nucleotides). We also determined that we can assemble and separate the complex from unbound proteins by documented methods, and showed that the complex is comparable in DNA replication activity after adding primase, DNA polymerase III holoenzyme and other components as the analogous complex formed on the corresponding circular ssDNA. Together with primer extension analysis, the results support a model that a molecule of DnaA bound to the DnaA box loads the DnaB-DnaC complex onto ssDNA at the right of the hairpin base to form the ABC complex. To understand helicase loading at the molecular level, we must identify the interacting surfaces of DnaA and DnaB. Our approach will employ HDX analysis of the ABC complex following a procedure already established in our lab. In companion experiments, we will determine the cryo-EM structure combined with 3D image reconstruction of the ABC complex, which will provide and understanding of the relative positions and orientations of DnaA, DnaB and DnaC in the complex. This information is essential to understand the mechanism of helicase loading. The 3D reconstruction will incorporate structural homology models of E. coli DnaA and the E. coli DnaB-DnaC complex using X-ray structures of G. stearothermophilus DnaB and the AAA+ domain of G. kaustophilus DnaA as templates.
Impacts
What was accomplished under these goals?
DnaB must form a complex with DnaC in order for this complex to load at oriC. Without knowing the interacting surfaces of DnaC and DnaB, we cannot understand why the DnaB-DnaC complex must form as a prerequisite for helicase loading, and how or whether this interaction leads to opening of the DnaB ring. Moreover, the mechanism underlying the transient interaction of primase with DnaB is poorly understood. The objectives of Aim 1 and 4 were to address these issues using the technique of hydrogen/deuterium exchange (HDX) together with mass spectrometry. This powerful method takes advantage of static X-ray crystallographic or NMR protein structures in interpreting HDX results that led to the provisional identification of sites in DnaB and DnaC that physically interact in forming the DnaB-DnaC complex. As verification, we constructed mutants bearing alanine substitutions in these sites. Their characterization together with the results above and molecular modeling underpin a structural model of the DnaB-DnaC complex. Dr. A. Daniel Jones, an authority on the methods of mass spectrometry including HDX, and Dr. Michael Feig, an expert in molecular modeling, were invaluable collaborators on this work. These findings are highly original, and very important to the field. The accepted view of DnaB was that its molecular structure of a closed ring was static. Our other results dramatically alter this paradigm. We discovered that DnaB alternates between an open and closed ring, and that the binding of DnaC to sites in the CTD of DnaB captures it as an open ring (Aim 1 objective). Our findings raise the possibility that ring molecules such as Lsm, Hfq, lamda exonuclease and Rad52 rely on the inherent ability to open and close in binding to RNA or DNA. In support, studies of the b clamp and T4 gp45 show that these ring molecules naturally open and close. Our integration of nucleic acid biochemistry and hydrogen/deuterium exchange of macromolecular complexes is an original approach to study the dynamic interactions of proteins during replication initiation, and may have encouraged others to use this combination of experimental approaches. For example, HDX analysis of S. solfataricus MCM identified an exterior surface that presumably interacts with the excluded ssDNA during unwinding. The goal of Aim 4 was to understand the dynamic interplay of DnaC and primase with DnaB during the transition from the stage of replication to the elongation phase of DNA replication. Earlier work showed that the interaction of primase with DnaB in the DnaB-DnaC complex at oriC, and primer formation lead to both the release of DnaC and DnaB activation. After the DnaB-DnaC complex has loaded at oriC, DnaB apparently assumes a conformation that is accessible to primase. On the basis that primase is known to interact with the NTD of DnaB, our HDX work, and unpublished results, which showed that DnaC bound to DnaB changes the conformation of the NTD of DnaB, we hypothesized that DnaC controls the ability of DnaB to interact with primase. This idea is a breakthrough because it was unexpected. In recent work (Felczak, M.M., Chodavarapu, S., and Kaguni, J.M., manuscript in preparation), we describe experiments showing that the binding of DnaC to DnaB occludes its NTD from primase. These studies underscore the dynamic interplay of DnaC and primase with DnaB. The objectives of Aim 3 was to test the hypothesis that the binding of DnaC to single-stranded DNA is necessary for DnaC function. We recently described the genetic selection of novel dnaC mutations. Speculating that two mutants that respectively carry F231S and W233L substitutions are defective in ssDNA binding, we showed that both bind aberrantly to ssDNA, but retain their ability to interact with DnaB. The significant finding is that the mutants are thermolabile in both helicase loading and ATP binding. Compared with studies showing that ATP is not needed for DnaC to form a complex with DnaB, they fail to clarify its critical role. Our study underscores the importance of ATP binding by DnaC during helicase loading. The regulatory inactivation of DnaA (RIDA) is a major mechanism that regulates the frequency of replication initiation. In RIDA, the Hda-b clamp complex bound to DNA directly interacts with ATP-bound DnaA (DnaA-ATP) to stimulate ATP hydrolysis. As DnaA-ATP is active in initiation but DnaA-ADP is not, this mechanism controls the activity of DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. In a collaborative study with two other labs, we determined the crystal structure of the Hda-β clamp complex (Kim, J.S., Nanfara, M.T., Chodavarapu, S., Jin, K.S., Babu, V.M.P, Ghazi, M.A., Kaguni, J.M., Sutton, M.D., and Cho, Y., manuscript submitted). This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp. One Hda surface containing the conserved clamp-binding motif (QLSLF) interacts with the β clamp cleft that is also recognized by other proteins whereas the second is a novel interface. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. In combination with the octameric structure of the Hda-b clamp complex, our mutational analyses of this complex indicate that its formation blocks the interaction of Hda with DnaA. Formation of the octameric complex is a novel mechanism that may regulate Hda function. The b sliding clamp plays a pivotal role in coordinating the actions of several proteins involved in DNA replication, repair and damage tolerance. Consistent with this important function, an elevated level of the b clamp slows growth by impeding DNA replication, possibly by sequestering one or more essential replication proteins away from the fork. Recently, several mutant forms of the b clamp, which failed to impede growth when their cellular abundance was increased, were shown to confer resistance to hydroxyurea when they were at the chromosomally encoded level. Under similar conditions, the wild type clamp did not confer hydroxyurea resistance. As hydroxyurea is a specific inhibitor of ribonucleotide reductase, these observation suggest that the mutant b clamps lead to the increased abundance of ribonucleotide reductase. In collaboration with another lab, we confirmed this expectation, showing that a mutant b clamp bearing an E202-to-K substitution led to an elevated level of ribonucleotide reductase relative to an isogenic wild type strain (Babu, V.M.P., Chodavarapu, S., Nanfara, M.T., Maul, R.W., Kaguni, J.M., and Sutton, M.D., manuscript in preparation). The increased abundance of ribonucleotide reductase was dependent on both hda and oriC functions. As described above, Hda acts together with the b clamp to stimulate the hydrolysis of ATP bound to DnaA. In addition, DnaA-ATP but not DnaA-ADP represses transcription of nrdAB, which encode the subunits of ribonucleotide reductase. Together, these findings support the model that Hda outcompetes with DNA polymerase III for binding to bE202K clamps loaded at oriC to catalyze more frequent RIDA. We also determined that the growth defect conferred by overexpression of the b clamp was suppressed by simultaneous overexpression of Hda, suggesting that elevated levels of the clamp impede growth by sequestering Hda to limit RIDA. Because bE202K supported increased levels of RIDA in vivo, this mutant clamp failed to impede E. coli growth when overexpressed. Our results are discussed in terms of models in which the b clamp coordinates the actions of Pol III in high fidelity processive DNA replication with those of Hda in RIDA.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Felczak, M.M., Sage, J.M., Hupert-Kocurek, K., Aykul, S., and Kaguni, J.M. (2016) Substitutions of conserved residues in the C-terminal region of DnaC cause thermolability in helicase loading. J Biol Chem. 291:4803-4812. doi: 10.1074/jbc.M115.708586. Epub 2016 Jan 4. PMID: 26728455
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Chodavarapu, S., and Kaguni, J.M. (2016) Replication initiation in bacteria. Enzymes 39, 1-30. doi: 10.1016/bs.enz.2016.03.001. Epub 2016 Apr 20. PMID: 27241926
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Kim, J.S., Nanfara, M.T., Chodavarapu, S., Jin, K.S., Babu, V.M.P, Ghazi, M.A., Kaguni, J.M., Sutton, M.D., and Cho, Y., Dynamic assembly of Hda and the sliding clamp in regulation of replication licensing. manuscript submitted.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chodavarapu, S., Jones, A.D., Feig, M., and Kaguni, J.M. (2016) DnaC traps DnaB as an open ring and remodels the domain that binds primase. Nucleic Acids Res. 44:210-220. doi: 10.1093/nar/gkv961. Epub 2015 Sep 29. PMID: 26420830
|
Progress 01/01/15 to 09/30/15
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 manuscripts by Chodavarapu et al. and Felczak and Kaguni. The work leading to these manuscripts is part of their training and professional development. How have the results been disseminated to communities of interest?The manuscripts will be published in "open access" journals. These articles may a;sp 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 complete experiments to show that 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 the ability of DnaB to interact with primase. Another series of experiments will use cryoelectron microscopy to characterize the structure of DnaA, DnaB and DnaC assembled at a DnaA box sequence. The purpose of this work 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. A critical event during replication initiation involves loading the DNA helicase at a DNA replication origin, which often requires the dynamic interactions between the DNA helicase and an accessory protein. In Escherichia coli, the DNA helicase is DnaB and DnaC is its loading partner. We used the method of hydrogen/deuterium exchange mass spectrometry to address the importance of DnaB-DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB's C-terminal domain to trap it as an open ring. This event correlates with conformational changes of the RecA fold of DnaB that is involved in nucleotide binding, and of the AAA+ domain of DnaC. DnaC also causes an alteration of the helical hairpins in the N-terminal domain of DnaB, presumably occluding this region from interacting with primase. Hence, DnaC controls the access of DnaB by primase. A manuscript describing this work has been submitted, and is under revision. In a separate manuscript in preparation, we show that primase interacts with DnaB but not when it is bound by DnaC. Hence, the binding of DnaC to DnaB leads to occlusion of the site in DnaB recognized by primase. The DnaB-DnaC complex binds to the unwound region of DNA within the Escherichia coli replication origin in an early step of helicase loading, but the biochemical events that lead to the stable binding of this complex to DNA are unclear. In other studies, we investigated the role of binding of DnaC to single-stranded DNA in DNA replication. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions near the C-terminus of DnaC (residue 245) reveals that they are active in DNA replication at 30oC, but are only partially active at 37oC. As the mutants remain able to interact with DnaB, their reduced activity at 37oC is not explained by their defective interaction with DnaB. By UV crosslinking, we show that DnaB greatly stimulates the binding of wild type DnaC to single-stranded DNA. Compared with DNA binding by wild type DnaC in the absence of DnaB, these mutants show an increased affinity for DNA as measured by UV crosslinking and by surface plasmon resonance, indicating that the mutations unmask this latent function of DnaC. This enhanced affinity correlates with defective DNA replication of the mutants. Together, these observations show that proper DNA binding by DnaC as a component of the DnaB-DnaC complex is critical for DNA replication. A manuscript describing this work has been submitted, and is under revision.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Chodavarapu, S., Jones, A.D., Feig, M., and Kaguni, J.M. 2015. DnaC traps DnaB as an open ring and remodels its N-terminal domain that binds primase.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Felczak, M.M., Sage, J.M., Hupert-Kocurek, K., Aykul, S., and Kaguni, J.M. 2015. The C-terminal region of DnaC controls its binding to single-stranded DNA. (manuscript under revision)
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