Double-strand DNA breaks (DSBs) occur in all cells multiple times per day. Homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ) are the two primary pathways for repairing DSBs. However, NHEJ is the more general pathway because it does not require a homologous donor, and NHEJ is sufficiently flexible that it can join almost any pair of incompatible DNA ends. The flexibility of NHEJ is essential because natural causes of DSBs (ionizing radiation, free radicals, enzyme malfunction) generate DNA ends with diverse chemical and structural configurations. Hence, that flexibility is well-suited for the task, but the price that we pay for that flexibility is that NHEJ causes DNA sequence changes at most sites where it functions (in humans and other vertebrates). Hence, NHEJ generates somatic cell mutations that cause cancer and likely contribute to aging. The flexibility of NHEJ represents one of the most sophisticated protein:DNA interaction pathways. Though we know most of the proteins that participate in NHEJ and know, in broad terms, what they do, we do not have a clear picture of how they function together, their spatial configuration, or the temporal relationships. With a clearer mechanistic and structural picture of human NHEJ, we will be in a position to develop small molecule inhibitors that may be useful for treating cancer (as chemotherapy or as a radiation sensitizer).
Aims 1 and 2 of this proposal focus precisely on the issues of structural and spatial relationships among the NHEJ proteins and with the DNA ends.
Aim 3 tests the hypothesis that the NHEJ ligase complex is important for making key end-to-end contacts during NHEJ.
Aim 4 describes development of a more robust NHEJ reconstitution.
In Aim 4, that system is then used to identify any remaining NHEJ factors by testing for enhancement of activity.
Aim 5 describes analysis of how histone octamers influence the nuclease, polymerase, and ligase activities of NHEJ. These studies will markedly improve our understanding of how NHEJ participates in processes such as chromosomal translocations that are of key importance in cancer etiology and how NHEJ might be made 'druggable'for therapeutic purposes.

Public Health Relevance

This project would clarify our insights into one of the key pathways for the repair of DNA damage. These insights will allow us to understand how some of the DNA damage that causes cancer and aging occurs. The benefits of some chemotherapy and therapeutic radiation work can be increased by this deeper level of understanding of this key DNA repair pathway. This project would clarify our insights into one of the key pathways for the repair of DNA damage. These insights will allow us to understand how some of the DNA damage that causes cancer and aging occurs. The benefits of some chemotherapy and therapeutic radiation work can be increased by this deeper level of understanding of this key DNA repair pathway.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01CA100504-11
Application #
8709816
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Pelroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Southern California
Department
Pathology
Type
Schools of Medicine
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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Reid, Dylan A; Keegan, Sarah; Leo-Macias, Alejandra et al. (2015) Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair. Proc Natl Acad Sci U S A 112:E2575-84
Lu, Zhengfei; Pannunzio, Nicholas R; Greisman, Harvey A et al. (2015) Convergent BCL6 and lncRNA promoters demarcate the major breakpoint region for BCL6 translocations. Blood 126:1730-1
Li, Sicong; Chang, Howard H; Niewolik, Doris et al. (2014) Evidence that the DNA endonuclease ARTEMIS also has intrinsic 5'-exonuclease activity. J Biol Chem 289:7825-34
Pannunzio, Nicholas R; Li, Sicong; Watanabe, Go et al. (2014) Non-homologous end joining often uses microhomology: implications for alternative end joining. DNA Repair (Amst) 17:74-80
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Zhang, Zheng Z; Pannunzio, Nicholas R; Hsieh, Chih-Lin et al. (2014) The role of G-density in switch region repeats for immunoglobulin class switch recombination. Nucleic Acids Res 42:13186-93
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Li, Sicong; Kanno, Shin-ichiro; Watanabe, Reiko et al. (2011) Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3' exonuclease and can participate in DNA end joining in a biochemical system. J Biol Chem 286:36368-77
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