For more than three decades, research supported by this grant has had a major impact on our understanding of how cells respond to damage to their DNA. It has made particularly important contributions to our knowledge of bacterial SOS responses to DNA damage and the crucial role that translesion DNA polymerases play in DNA damage tolerance and mutagenesis. The proposed research addresses critical problems concerning a newly identified mechanism of stress-induced lethality that results from the incorporation of oxidized deoxynucleotides into DNA, the regulation and function of translesion DNA polymerases, and NusA-dependent mechanisms that couple transcription elongation to DNA repair and damage tolerance. In a completely unanticipated development, our recent research has provided evidence that the incorporation of oxidized deoxynucleotides into DNA contributes significantly to the death of bacterial cells undergoing certain types of stress, which include DinB overproduction, bactericidal antibiotics, and expression of a MalE'-'LacZ fusion protein. This poorly understood type of lethality appears to be directly relevant to cancer and various human diseases. We will carry out in vivo and in vitro experiments to test our model that death results from double stand breaks caused by incomplete base excision repair of nearby incorporated oxidized nucleotides. These should provide important insights to its mechanism, significance, and generality. We have discovered that Y Family DinB-related to DNA polymerases have a striking ability to preferentially carry out accurate translesion synthesis (TLS) over certain adducts typified by N2-furfuryl-dG. We have termed these stealth lesions since the high ratio of DinB to replicative polymerase makes them largely invisible to DNA replication, but they remain in the DNA to cause problems with transcription. Our proposed experiments will yield new mechanistic information about how the dynamic switching between replicative and TLS DNA polymerases at such a lesion is regulated. They will also extend our understanding of the nature of such stealth lesions and of how this capability for specialized bypass relates to DinB's other in vitro and in vivo roles. We have discovered unanticipated roles in DNA repair and damage tolerance for NusA, a component of elongating RNAP polymerases. Our observations have led us to propose a previously unrecognized pathway of NusA-dependent transcription-coupled repair (TCR) that is of particular importance for the removal of stealth lesions from the transcribed strand of expressed genes. Other evidence has led us to suggest a model for NusA-dependent transcription-coupled TLS (TC-TLS) that can help cells deal with transcriptional problems created by gaps in the transcribed strand that result from lesions in the non-transcribed strand. Our proposed experiments will offer new insights to how NusA helps cells repair or tolerate DNA damage.

Public Health Relevance

The proposed research will offer insights into the mechanism and regulation of key fundamental processes that enable cells to repair and tolerate damage to their genetic material. These processes are responsible for the mutations that lead to cancer, while manipulation of these processes can make chemotherapy more effective. Investigation of a newly discovered mechanism of cell death dependent on the incorporation of oxidized building blocks into DNA should help lead to new ways to treat cancer and possibly to the design of new classes of antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA021615-40
Application #
9253352
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Okano, Paul
Project Start
1991-02-01
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
40
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Gruber, Charley C; Walker, Graham C (2018) Incomplete base excision repair contributes to cell death from antibiotics and other stresses. DNA Repair (Amst) :
Takahashi, Noriko; Gruber, Charley C; Yang, Jason H et al. (2017) Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality. Proc Natl Acad Sci U S A :
Dwyer, Daniel J; Collins, James J; Walker, Graham C (2015) Unraveling the physiological complexities of antibiotic lethality. Annu Rev Pharmacol Toxicol 55:313-32
Belenky, Peter; Ye, Jonathan D; Porter, Caroline B M et al. (2015) Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage. Cell Rep 13:968-80
Pandey, Shree P; Winkler, Jonathan A; Li, Hu et al. (2014) Central role for RNase YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria. BMC Genomics 15:121
Penterman, Jon; Singh, Pradeep K; Walker, Graham C (2014) Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa. J Bacteriol 196:3351-9
Dwyer, Daniel J; Belenky, Peter A; Yang, Jason H et al. (2014) Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci U S A 111:E2100-9
Shrivastav, Nidhi; Fedeles, Bogdan I; Li, Deyu et al. (2014) A chemical genetics analysis of the roles of bypass polymerase DinB and DNA repair protein AlkB in processing N2-alkylguanine lesions in vivo. PLoS One 9:e94716
Opperman, Timothy J; Kwasny, Steven M; Kim, Hong-Suk et al. (2014) Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli. Antimicrob Agents Chemother 58:722-33
Kath, James E; Jergic, Slobodan; Heltzel, Justin M H et al. (2014) Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis. Proc Natl Acad Sci U S A 111:7647-52

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