Progress 04/01/14 to 03/31/19
Outputs
Target Audience:Other scientists working in the broad area of DNA repair., Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results have been published and presented at scientific meetings. 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.Aim 1. How does DNA-PKcs autophosphorylation facilitate c-NHEJ.An over-riding goal of this application was to determine the mechanistic basis of how autophosphorylation of DNA-PKcs facilitates c-NHEJ. Using a mutational approach, we determined that DNA-PK was activated in a step-wise manner such that two separate phosphorylation events, that occur at two clusters of phosphorylation sites reciprocally regulate access of DNA ends to end processing enzymes. Thus, if phosphorylation in one cluster is blocked (by mutational strategies), end-processing is severely limited, whereas if phosphorylation is blocked at the other cluster, end-processing is markedly enhanced. Moreover, blocking sites that facilitate end-processing results in a dominant negative like effect on cell survival after exposure to agents that induce DNA double strand breaks (DSBs). Finally, the dominant negative effect is absolutely dependent on kinase activity of the enzyme, and the capacity to phosphorylate the reciprocal cluster.In sum, these data suggest a step-wise mode of kinase function. In a second series of experiments to address how DNA-PKcs autophosphorylation facilitate c-NHEJ, an in vitro approach was utilized that was designed to assess whether DNA-PK was activated in cis or trans. Using pull-down approaches, we made the surprising observation that DNA-PKcs could be activated - independently of DNA ends, and independently from Ku, by restraining the enzyme at its N-terminus, suggesting an allosteric mechanism of activation. During the course of this RO1, a high resolution crystal structure of DNA-PKcs was published (Blundell and colleagues). The major conclusion from their study was that DNA-PK is activated by an allosteric mechanism, in complete agreement with our model. Their study also confirmed our conclusion that whereas one major autophosphorylation cluster was located in a disordered region of the molecule, the second major cluster was likely proximate to the DNA binding pocket. A recentreportofa mouse model, bearing a kinase inactive version of DNA-PKcs that had a much moremore severe phenotype than cell strains expressing kinase inactive DNA-PKcs. This represented a major discrepancy in the published literature.We put forth a significant effort to clarify these discrepancies. Our study suggests that loss of ATM in DNA-PKcs deficient cells or animals, but not in cells or animals expressing kinase inactive DNA-PKcs likely impacts the phenotype observed in mice. Moreover, our data suggest that the more severe impact of inactive DNA-PKcs versus DNA-PKcs ablation, can only be appreciated in non-transformed cell strains. Finally, significant controversy exists as to whether certain sites in DNA-PKcs are autophosphorylated, or targeted by either ATM or ATR.Our data suggest that DNA-PKcs is predominately autophosphorylated. We also studied how specific phosporylations that occur during VDJ recombination affect how RAG mediated DNA double strand breaks are repaired. Briefly, unlike most DNA-PKcs deficient mouse cell strains, we show here that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those studied here) and in DNA-PKcs deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the "classical" non-homologous end-joining pathway. We extended these results and utilized this system to perform a structure/function analysis of how DNA-PK and ATM affect joining of RAG mediated DSBs.. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM. B.What is the role of DNA-PK and c-NHEJ during replication stress?Although DNA-PK is primarily considered a c-NHEJ factor, evidence continues to mount documenting how DNA-PK affects other repair pathways.There is good evidence that replication stress is a predominant source of genomic instability. DNA-PK can access and is activated by single DNA double-stranded ends that result from replication fork collapse.; however, c-NHEJ joining of these single double-stranded ends would generate potentially pathogenic genomic alterations.So, one might think that disrupting c-NHEJ during replication stress would be beneficial; paradoxically, cultured cells that lack DNA-PK or other c-NHEJ components are more sensitive to replication stress induced by hydroxyurea than are isogenic, c-NHEJ proficient controls. The central question of Specific Aim 2 is why is c-NHEJ important for survival after replication stress? We have collaborated with Dr. Thomas Helleday's laboratory in Sweden and demonstrated thatDNA-PKcs and PARP1 bind to unresected stalled DNA replication forks where they recruit XRCC1 to facilitate repair. C.Do XRCC4/XLF filaments function to bridge DNA in living cells, and how does DNA-PK regulate their function?XRCC4's role in stabilizing and promoting DNA ligase IV stability and activity is well characterized. More recently, XLF was discovered as an XRCC4 interacting protein that also functions to facilitate ligase IV function. We have shown that XRCC4/XLF filaments bridge DNAin vitroand promote end joiningin vivo. Our previous work demonstrating that XRCC4 mutants that do not form filaments only partially reverse the radiosensitive and end joining deficient phenotypes of XRCC4 deficient cells, suggest that these filaments are functionally relevant in living cells.We also characterized two XLF mutants that do not interact with XRCC4, andcannotform filaments or bridge DNA.Onemutant is fully sufficient in stimulating ligation byXRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements thec-NHEJdeficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for DNA bridging.To determine whetherthelack of XRCC4/XLF bridging might be compensated for by other factors, candidate repair factors were disrupted in XLF-or XRCC4-deficient cells.The lossof either ATM or the newly described XRCC4/XLF-like factor, PAXX,accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss) this reflects a greater requirement for XRCC4/XLF bridging.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Neal, J.A. and Meek, K. Deciphering phenotypic variance in different models of DNA-PKcs deficiency. DNA Repair. 73:7-16. 2019.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: The target audience for this research are other scientists working in the areas of DNA repair, cancer therapy, immune system development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provides training opportunities for three undergraduate students in my laboratory, and provides the major research project for one post-doctoral fellow. How have the results been disseminated to communities of interest?The work has been published. What do you plan to do during the next reporting period to accomplish the goals?In the last few years, the laboratory has focused on how G-quadruplex binding ligands induce DNA damage; we find that one G-quadruplex binding ligand induces robust DNA double strand breaks that require the classical non-homologous end joining pathway for repair. Surprisingly, expression of high levels of DNA polymerase theta, a major factor in the alternative non-homologous end joining pathway actually sensitizes cells to this G-quadruplex binding ligand. We are investigating the mechanism. We are continuing our previous work on understanding how DNA-PK affects the integrity of VDJ recombination.Although we have deciphered how ATM, DNA-PK, and the RAG complex cooperate to restrict signal end joining to the NHEJ pathway, we are still claryifying additional effects on coding end joining. Moreover, experiments are underway to discern biochemical basis for this effect. Finally, experiments are underway to test this model on chromosomal recombination using first a cultured B cell line that undergoes inducible robust VDJ recombination. This strategy is designed so that we can also assess chromosomal recombination in living animals.
Impacts
What was accomplished under these goals?
My laboratory focuses on why DNA-PK's kinase activity is required during non-homologous DNA end joining. The fact that kinase activity is requisite for function implies that phosphorylation by DNA-PK activates a downstream factor. How DNA-PK functions in the broader context of DNA double strand break repair is of considerable interest. More than a decade ago, my laboratory demonstrated that inactivating the enzyme by a mutational approach, completely abrogated DNA-PK's function; however, the catalytically inactive enzyme did not impart any detectable dominant negative effect in the cell culture model utilized in our experiments. To our surprise, a recent report documents a potential strong dominant negative effect of a kinase inactivating mutation in DNA-PKcs when introduced into the mouse germline. In the last year, we have focused significant effort in clarifying these discordant results. We have shown that kinase inactivatingDNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity.Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we demonstratedthat DNA-PK primarily autophosphorylates. In recent years, my laboratory has focused on XRCC4 and XLF, two factors previously thought to function only in the final ligation step of DNA repair. Structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XRCC4/XLF might stimulate ligation. Last year we provided compelling data that XRCC4/XLF filaments are regulated by DNA-PK phosphorylation. Moreover, we showed that the phosphorylation sites involved are functionally redundant with one another. This data was published earlier this year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Normanno, D., N�grel, A., de Melo, A.J., Betzi, S., Meek, K., and Modesti, M. Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining. E.Life. 6.pii: e22900. PMID 28500754 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
M�ller, T.A., Struble, S.L., Meek, K., Hausinger, R.A. Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells. BBRC. 495(1):98-103. 2018
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Neal, J.A. and Meek, K. Deciphering phenotypic variance in different models of DNA-PKcs deficiency. DNA Repair. Submitted. 2018.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this research are other scientists working in the areas of DNA repair, cancer therapy, immune system development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?This work has been published. What do you plan to do during the next reporting period to accomplish the goals?Although we have deciphered how ATM, DNA-PK, and the RAG complex cooperate to restrict signal end joining to the NHEJ pathway, we are still claryifying additional effects on coding end joining. Moreover, experiments are underway to discern biochemical basis for this effect. Finally, experiments are underway to test this model on chromosomal recombination using first a cultured B cell line that undergoes inducible robust VDJ recombination. This strategy is designed so that we can also assess chromosomal recombination in living animals.
Impacts
What was accomplished under these goals?
Outputs. My laboratory focuses on why DNA-PK's kinase activity is required during non-homologous DNA end joining. The fact that kinase activity is requisite for function implies that phosphorylation by DNA-PK activates a downstream factor. How DNA-PK functions in the broader context of DNA double strand break repair is of considerable interest. Although most DNA double strand breaks (DSBs) can be repaired by either the non-homologous end joining (NHEJ) pathway or by homologous recombination (HR), DSBs introduced during VDJ recombination, a programed DNA recombination mechanism that is required for immune system development, are repaired exclusively by NHEJ. We have been exploring the mechanistic basis for this bias. Our data suggest that a related protein kinase, ATM (Ataxia Telangiectasia Mutated) cooperates with DNA-PK and the immune specific nuclease, the RAG (Recombination Activating Gene) complex to guide DSBs directly into the NHEJ complex. Although the evidence is overwhelming and unequivocal that ATM contributes to accurate non-homologous end joining of VDJ coding segments, and restrains their participation in genomic translocations, a clear mechanistic understanding of how ATM actually does this job is lacking. Briefly, the ATM kinase is central to the DNA damage response; it follows that this large kinase is central to regulating how developing lymphocytes respond to their self-imposed DNA damage during VDJ recombination. Recently, we found that ATM ablation in many cultured cell strains results in increased VDJ joining in episomal assays, a completely counter-intuitive result if ATM were to have a direct functional role in end joining. We considered that if ATM's role was instead, to regulate the RAG post cleavage complex(s), loss of ATM might result in increased release of VDJ recombination intermediates and more rapid joining, explaining the increased recombination observed. We fine-tuned the assay so that a structure/function, reductionist approach could be employed to delineate if and how ATM directly affects the RAG complex. We find that ATM limits both signal and coding end joining. This inhibition is specific for VDJ end joining; joining of other DSBs on episomal substrates is not affected. ATM's catalytic activity, and the non-core C-termini of both RAG1 and RAG2, as well as potential phosphorylation targets in RAG2's C-terminus, are required for ATM's capacity to limit signal (but not coding) end joining. Although not unequivocal, our preliminary data are the most compelling to date implicating a direct effect of DNA-PK/ATM phosphorylation of RAG2's non-catalytic domain in modulating the stability of the RAG post cleavage complex. Data from my laboratory published in the last two years has provided strong structure function data that help understand this mechanistically. Moreover, emerging data from other laboratories provides strong in vivo support for this model. In recent years, my laboratory has focused on XRCC4 and XLF, two factors previously thought to function only in the final ligation step of DNA repair. Structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XRCC4/XLF might stimulate ligation. Last year we provided compelling data that XRCC4/XLF filaments are regulated by DNA-PK phosphorylation. Moreover, we showed that the phosphorylation sites involved are functionally redundant with one another. This data was published earlier this year. In collaboration with the Modesti lab in Marseille, we were the first group to demonstrate that XLF and a newly described NHEJ factor, PAXX are functionally redundant with each other. This year, three high profile publications corroborated this result. This year we demonstrated that PAXX is actually an accessory factor during NHEJ. This data was published earlied this year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Masani, S., Han, L, Meek, K., and Yu, K. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. U.S.A. 113:1261-1266. 2016.
Neal, J.A., Xu, Y., Abe, M., Hendrickson, E., and Meek, K. Restoration of ATM expression in DNA-PKcs deficient cells inhibits signal end joining. J. Immunol. 150:1654. 2016.
Meek, K., Xu, Y., Bailie, C., Yu, K., and Neal, J.A. The ATM kinase restrains joining of both VDJ signal and coding ends. J. Immunol. 197:3165-3174, 2016.
Tadi, S.K., Tellier-Leb�gue, C., Nemoz, C., Drevet, P., Audebert, S., Roy, S., Meek, K., Charbonnier, J.B., and Modesti, M. PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF. Cell Reports.17:541-555. 2016
Normanno, D., N�grel, A., de Melo, A.J., Betzi, S., Meek, K., and Modesti, M. Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining. E.Life. 6.pii: e22900. PMID 28500754 2017.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience for this research are other scientists working in the areas of DNA repair, cancer therapy, immune system development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research has also provided undergraduate research projects for four undergraduate students working in my laboratory over the past year. How have the results been disseminated to communities of interest?The results of this research are disseminated by Peer-review publication (listed above). In addition, these results were presented at an international Abcam, "Maintenance of Genomic Instability" March, Panama City, Panama. What do you plan to do during the next reporting period to accomplish the goals?Although we have deciphered how ATM, DNA-PK, and the RAG complex cooperate to restrict signal end joining to the NHEJ pathway, we are still claryifying additional effects on coding end joining. Moreover, experiments are underway to discern biochemical basis for this effect. The second aim of this study is to dissect how NHEJ and particularly DNA-PK protect genomic integrity during replication stress. This question will be a central focus in the next funding period.
Impacts
What was accomplished under these goals?
My laboratory focuses on why DNA-PK's kinase activity is required during non-homologous DNA end joining. The fact that kinase activity is requisite for function implies that phosphorylation by DNA-PK activates a downstream factor. How DNA-PK functions in the broader context of DNA double strand break repair is of considerable interest. Although most DNA double strand breaks (DSBs) can be repaired by either the non-homologous end joining (NHEJ) pathway or by homologous recombination (HR), DSBs introduced during VDJ recombination, a programed DNA recombination mechanism that is required for immune system development, are repaired exclusively by NHEJ. We have been exploring the mechanistic basis for this bias. Our data suggest that a related protein kinase, ATM (Ataxia Telangiectasia Mutated) cooperates with DNA-PK and the immune specific nuclease, the RAG (Recombination Activating Gene) complex to guide DSBs directly into the NHEJ complex. Although the evidence is overwhelming and unequivocal that ATM contributes to accurate non-homologous end joining of VDJ coding segments, and restrains their participation in genomic translocations, a clear mechanistic understanding of how ATM actually does this job is lacking. Data from my laboratory published this year provide strong structure function data that help understand this mechanistically. Briefly, the ATM kinase is central to the DNA damage response; it follows that this large kinase is central to regulating how developing lymphocytes respond to their self-imposed DNA damage during VDJ recombination. Recently, we found that ATM ablation in many cultured cell strains results in increased VDJ joining in episomal assays, a completely counter-intuitive result if ATM were to have a direct functional role in end joining. We considered that if ATM's role was instead, to regulate the RAG post cleavage complex(s), loss of ATM might result in increased release of VDJ recombination intermediates and more rapid joining, explaining the increased recombination observed. We fine-tuned the assay so that a structure/function, reductionist approach could be employed to delineate if and how ATM directly affects the RAG complex. We find that ATM limits both signal and coding end joining. This inhibition is specific for VDJ end joining; joining of other DSBs on episomal substrates is not affected. ATM's catalytic activity, and the non-core C-termini of both RAG1 and RAG2, as well as potential phosphorylation targets in RAG2's C-terminus, are required for ATM's capacity to limit signal (but not coding) end joining. Although not unequivocal, our preliminary data are the most compelling to date implicating a direct effect of DNA-PK/ATM phosphorylation of RAG2's non-catalytic domain in modulating the stability of the RAG post cleavage complex. In recent years, my laboratory has focused on XRCC4 and XLF, two factors previously thought to function only in the final ligation step of DNA repair. Structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XRCC4/XLF might stimulate ligation. By studying a panel of XLF and XRCC4 mutants that cannot with each other, we have determined that the bridging function of XRCC4/XLF filaments is variably required in different cell types. The fact that we demonstrate that this DNA bridging is functionally relevant, intuits that XRCC4/XLF filaments play an early role during DNA repair. Several high impact publications described a third XRCC4/XLF paralogue, PAXX. In the previous funding period, we demonstrate that PAXX and XLF are functionally redundant; this finding was recently corroborated by two other laboratories; in the last funding period, in collaboration with the Modesti laboratory, we have delineated the mechanistic basis for the functional redundancy of PAXX and XLF.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ying, S., Chen, Z., Medhurst, A.L., Neal, J.A., Bao, Z., Mortusewicz, O., McGouran, J., Song, X., Shen, H., Hamdy, F.C., Kessler, B.M., Meek, K., Helleday, T. DNA-PKcs and PARP1 bind to unresected stalled DNA replication forks where they recruit XRCC1 to mediate repair. Cancer Res. 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Masani, S., Han, L, Meek, K., and Yu, K. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc. Natl. Acad. Sci. U.S.A. 113:1261-1266. 2016.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Neal, J.A., Xu, Y., Abe, M., Hendrickson, E., and Meek, K. Restoration of ATM expression in DNA-PKcs deficient cells inhibits signal end joining. J. Immunol. 150:1654. 2016.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Meek, K., Xu, Y., Bailie, C., Yu, K., and Neal, J.A. The ATM kinase restrains joining of both VDJ signal and coding ends. J. Immunol. In press 2016.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Tadi, S.K., Tellier-Leb�gue, C., Nemoz, C., Drevet, P., Audebert, S., Roy, S., Meek, K., Charbonnier, J.B., and Modesti, M. PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF. Cell Reports. In press. 2016
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience for this research are other scientists working in the areas of DNA repair, cancer therapy, immune system development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research in my laboratory has provided the basis for the graduate research project of a PhD student, Sunetra Roy who recently completed her PhD in Cellular & Molecular Biology. This research has also provided undergraduate research projects for four undergraduate students working in my laboratory over the past year. How have the results been disseminated to communities of interest?The results of this research are disseminated by Peer-review publication (listed above). In addition, these results were presented at an international Keystone Conference, "Genomic Instability and DNA Repair" March 1-6, Whistler, British Columbia, Canada. What do you plan to do during the next reporting period to accomplish the goals?Experiments are underway to address the specific aims of the proposal.
Impacts
What was accomplished under these goals?
My laboratory focuses on why DNA-PK's kinase activity is required during non-homologous DNA end joining. The fact that kinase activity is requisite for function implies that phosphorylation by DNA-PK activates a downstream factor. We have shown that DNA-PK's phosphorylation of several NHEJ factors is not functionally relevant, but it is becoming clear that DNA-PK itself, is the kinase's primary target. Autophosphorylation occurs on many sites (probably more than 30 of the 4129 residues), and is functionally complex. Previously we defined two major clusters of autophosphorylation sites that reciprocally regulate DNA end processing during NHEJ. We also identified and characterized an autophosphorylation site within the activation (or t) loop of the kinase domain; phosphorylation of this site regulates activity. Our current model is that via a series of autophosphorylation events, DNA-PK undergoes a series of conformational changes that facilitate each step of NHEJ. How DNA-PK functions in the broader context of DNA double strand break repair is of considerable interest. Although most DNA double strand breaks (DSBs) can be repaired by either the non-homologous end joining (NHEJ) pathway or by homologous recombination (HR), DSBs introduced during VDJ recombination, a programed DNA recombination mechanism that is required for immune system development, are repaired exclusively by NHEJ. We have been exploring the mechanistic basis for this bias. Our data suggest that a related protein kinase, ATM (Ataxia Telangiectasia Mutated) cooperates with DNA-PK and the immune specific nuclease, the RAG (Recombination Activating Gene) complex to guide DSBs directly into the NHEJ complex. Although this has been hypothesized for approximately 10 years, new data from my laboratory provide strong structure function data in support of this hypothesis. Two publications are under revision and in preparation describing these new findings. In recent years, my laboratory has focused on XRCC4 and XLF, two factors previously thought to function only in the final ligation step of DNA repair. Recent structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XRCC4/XLF might stimulate ligation. By studying a panel of XLF and XRCC4 mutants that cannot with each other, we have determined that the bridging function of XRCC4/XLF filaments is variably required in different cell types. The fact that we demonstrate that this DNA bridging is functionally relevant, intuits that XRCC4/XLF filaments play an early role during DNA repair. This finding requires a reexamination of the current dogma of a step by step progression of non-homologous end joining. We also have determined that other factors, specifically ATM and a newly described XRCC4/XLF-like factor, PAXX, can compensate for XRCC4/XLF bridging. Finally, (as with other c-NHEJ factors) some XLF deficient cells display increased sensitivity to replication stress; surprisingly neither XLF's ability to stimulate X4/L4 nor its ability to bridge DNA (with XRCC4) is required for this potential role for XLF in abating replication stress. These findings were published earlier this year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Roy, S., deMelo, A.J., Xu, Y., Tadi, S.K., Negrel, A., Hendrickson, E., Modesti, M., and Meek, K. XRCC4/XLF interaction is variably required for DNA repair and is not required for ligase IV stimulation. Mol. Cell Biol. 35:3017-3028. 2015.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Ying, S., Chen, Z., Medhurst, A.L., Bao, A., McGouran, J., Neal, J.A., Song, X., Shen, H., Hamdy, F.C., Kessler, B.M., Meek, K., Helleday, T. DNA-PKcs and PARP1 bind to unresected stalled forks and recruit XRCC1 to mediate replication repair. Cancer Res. Under revision. 2015.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Neal, J.A., Xu, T., Abe, M., Hendrickson, E. and Meek, K. Restoration of ATM expression in DNA-PKcs deficient cells impairs signal end joining and potentiates the radiosensitive phenotype. Under revision. 2015.
|
Progress 04/01/14 to 09/30/14
Outputs
Target Audience: The target audience for this research are other scientists working in the areas of DNA repair, cancer therapy, immune system development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The XRCC4/XLF project represents the PhD dissertation work of a graduate student, Sunetra Roy. The DNA-PK mutational study was conducted in large part by Dr. Jessica Neal, a research associate in my laboratory who has continued to develop into a superb scientist. She supervised three undergraduate students to assist her in these studies. Two of those students are planning applications to professional school (DVM and MD). How have the results been disseminated to communities of interest? These results have been (or are in the process of being reviewed) for scientific publication. In addition, these studies were presented by myself and by my student at an international conference "Maintenance of Genome Stability 2014" organized by Abcam (St. Kitts, 3/32014). What do you plan to do during the next reporting period to accomplish the goals? Experiments are being initiated to study DNA repair outcomes at precise lesions during replication stress. Mutational analyses of XRCC4/XLF filaments and DNA-PKcs in cultured cell strains is ongoing.
Impacts
What was accomplished under these goals?
My laboratory focuses on why DNA-PK's kinase activity is required during non-homologous DNA end joining. The fact that kinase activity is requisite for function implies that phosphorylation by DNA-PK activates a downstream factor. We have shown that DNA-PK’s phosphorylation of several NHEJ factors is not functionally relevant, but it is becoming clear that DNA-PK itself, is the kinase’s primary target. Autophosphorylation occurs on many sites (probably more than 30 of the 4129 residues), and is functionally complex. Previously we defined two major clusters of autophosphorylation sites that reciprocally regulate DNA end processing during NHEJ. We also identified and characterized an autophosphorylation site within the activation (or t) loop of the kinase domain; phosphorylation of this site regulates activity. Our current model is that via a series of autophosphorylation events, DNA-PK undergoes a series of conformational changes that facilitate each step of NHEJ. In the last funding period we have continued our work unraveling the functional complexities of DNA-PK's autophosphorylation. Our work has revealed that DNA-PK is actually targeted to a variety of lesions that are actually not usually repaired via NHEJ. Our data suggest that appropriate autophosphorylations are important to disengage NHEJ from DNA damage that cannot be repaired by NHEJ. This is particularly important in human cells that express roughly 50 times more DNA-PK than other vertebrates. By studying a panel of DNA-PKcs autophosphorylation mutants derived in my laboratory, we have proposed a new model explaining DNA-PK's function when bound to DNA ends. Briefly, our data, coupled with recent structural data suggest a model whereby early phosphorylations promote initiation of NHEJ; whereas phosphorylations, potentially located in a "hinge" region between the two major domains of DNA-PKcs, lead to regulated conformational changes that initially promote NHEJ, and eventually disengage NHEJ. In recent years, my laboratory has focused on XRCC4 and XLF, two factors previously thought to function only in the final ligation step of DNA repair. Recent structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XRCC4/XLF might stimulate ligation. By studying a panel of XLF and XRCC4 mutants that cannot with each other, we have determined that the bridging function of XRCC4/XLF filaments is variably required in different cell types. We also have determined that other factors, specifically ATM and a newly described XRCC4/XLF-like factor, PAXX, can compensate for XRCC4/XLF bridging. Finally, (as with other c-NHEJ factors) some XLF deficient cells display increased sensitivity to replication stress; surprisingly neither XLF's ability to stimulate X4/L4 nor its ability to bridge DNA (with XRCC4) is required for this potential role for XLF in abating replication stress.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Neal, J. A., Sugiman-Marangos, S., VanderVere-Carozza, P., Wagner, M., Turchi, J., Lees-Miller, S.P., Junop, M.S., and Meek, K. Unraveling the complexities of DNA-PK autophosphorylation. Mol Cel. Biol. 34:2162-75. 2014.
Douglas, P, Ye, R, Trinkle-Mulcahy, L, Neal JA, De Wever, V, Morrice, NA, Meek, K, Lees-Miller, SP. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis. Biosci Rep. 34. 2014.
Petersen-Jones, S., Meek K. DNA damage: offing KAP to stay focused in the dark. Curr Biol. 24:392-4. 2014.
Neal, J.A., Xu, Y., Abe, M., Hendrickson, E., and Meek, K. ATM and DNA-PK cooperate to restrict VDJ signal end repair to the c-NHEJ pathway. under review. 2014.
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