Project 5 ? Double-Strand Break Repair and Pathway Choice PROJECT SUMMARY/ABSTRACT DNA double strand breaks (DSBs) are the most deleterious form of DNA damage, and failure to repair DSBs leads to cell death or chromosomal instability that is a hallmark of cancer. DSB detection, signaling and initiation of one of three distinct DSB repair pathways are critical processes for the cell's fate and genomic integrity. Project 5 integrates into SBDR-4 by focusing on dynamic complexes acting in DSB Repair (DSBR) initiation and signaling, non-homologous end joining (NHEJ) progression, and pathway choice between homologous recombination repair (HRR) and NHEJ. DSBR is directly relevant to cancer biology and interventions. The impact of selecting between HRR, which is error-free, and NHEJ, which can be error-prone, depends upon cell cycle status and the nature of the damage. Current chemotherapy and radiotherapy create DSBs, and PARP inhibitors specifically kill cancer cells that are DSBR deficient through synthetic lethality. Furthermore, the specific targeting of cancer cell susceptibilities by selectively blocking specific DSBR responses provides a promising approach to kill cancer cells with less damage to normal cells. Yet, without a structural and mechanistic knowledge of DSB repair, our ability to interpret mutations arising from the clinic and to provide knowledge for improved design of future therapies is limited. Project 5 will characterize macromolecular interfaces, assemblies, conformations, and key phosphorylation and ubiquitination events underlying DSBR initiation, regulation and pathway choice.
In Aim 1, MRN-DNA interactions will be defined, and we will determine structures for MRN, CtIP and ATM activation and regulation. We will define their key roles in initiating HRR, to identify mutations and inhibitors to dissect multi-functionality and to uncover synthetic lethality.
In Aim 2, we will define NHEJ interfaces, focusing on the PNKP-XRCC4-LigIV complexes and the nuclease Artemis to define their critical roles in NHEJ.
In Aim 3 we will focus on the heart of pathway choice by determining how DNA-PK and MRN compete for DNA in the context of chromatin, and via the post- translational modifications of phosphorylation and ubiquitination that regulate NHEJ and HRR initiation and signaling. Project 5 requires extensive use of the EMB core for constructs and protein complex expression, and of the SCB Core for structural experiments. Project 5 will exchange mutants, constructs, inhibitors and other results with Projects 2-4 and collaborate to examine impacts on multiple DNA repair pathways with Project 1. The anticipated outcomes of the proposed experiments include an actionable mechanistic knowledge to inform the Cancer Genome Atlas and system level studies of knockouts. This knowledge will help to achieve NCI missions by bridging the gap from sequences and mutations to phenotype prediction by defining biologically validated atomic complexes, conformations, and mechanisms for synthetic lethality.

Public Health Relevance

Project 5 ? Double-Strand Break Repair and Pathway Choice PROJECT NARRATIVE Differences in the repair of DNA double strand breaks (DSBs), formed from failed DNA replication and from DNA damage by cancer therapies, contribute to differences in cancer predispositions and outcomes to therapies. In Project 5, we will define molecular mechanisms that signal and coordinate DSB repair steps, and how cells choose among different repair pathways. Project results will provide fundamental knowledge to advance cancer biology, to predict the impact of patient mutations, and to design more effective therapeutic approaches.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA092584-20
Application #
10003206
Study Section
Special Emphasis Panel (ZCA1)
Project Start
2001-09-27
Project End
2021-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
20
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Chavez, Diana A; Greer, Briana H; Eichman, Brandt F (2018) The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression. J Biol Chem 293:8484-8494
Wang, Jing L; Duboc, Camille; Wu, Qian et al. (2018) Dissection of DNA double-strand-break repair using novel single-molecule forceps. Nat Struct Mol Biol 25:482-487
Crickard, J Brooks; Kaniecki, Kyle; Kwon, Youngho et al. (2018) Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase. Proc Natl Acad Sci U S A 115:E10041-E10048
Syed, Aleem; Tainer, John A (2018) The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair. Annu Rev Biochem 87:263-294
Howes, Timothy R L; Sallmyr, Annahita; Brooks, Rhys et al. (2018) Erratum to ""Structure-activity relationships among DNA ligase inhibitors; characterization of a selective uncompetitive DNA ligase I inhibitor"" [DNA Repair 60C (2017) 29-39]. DNA Repair (Amst) 61:99
Bhattacharyya, Sudipta; Soniat, Michael M; Walker, David et al. (2018) Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase. Proc Natl Acad Sci U S A 115:E11614-E11622
Tsai, Chi-Lin; Tainer, John A (2018) Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes. Methods Enzymol 599:157-196
Ogorzalek, Tadeusz L; Hura, Greg L; Belsom, Adam et al. (2018) Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy. Proteins 86 Suppl 1:202-214
Langelier, Marie-France; Zandarashvili, Levani; Aguiar, Pedro M et al. (2018) NAD+ analog reveals PARP-1 substrate-blocking mechanism and allosteric communication from catalytic center to DNA-binding domains. Nat Commun 9:844
Crickard, J Brooks; Greene, Eric C (2018) Biochemical attributes of mitotic and meiotic presynaptic complexes. DNA Repair (Amst) :

Showing the most recent 10 out of 484 publications