Cancer is a genetic disease caused by mutations acquired in somatic cells. A main driving force behind this mutation is chemical alterations in DNA resulting from cell-derived processes and exogenous DNA damaging agents. A paradox is that the repair systems that normally reverse these DNA lesions must also be responsible for transforming them into heritable sequence alterations. Thus, malignancy is a result of DNA repair failure. The double-stranded chromosome break is a DNA lesion of particular importance as it gives rise to the chromosomal rearrangements that are nearly ubiquitous in cancer. Cells possess two mechanistically distinct pathways of double-strand break repair, homology-directed repair and nonhomologous end joining (NHEJ). The long-term objective of this proposal is to understand how the balance between these two pathways is maintained and might therefore be perturbed during mutagenesis, which first requires a detailed understanding of their mechanisms. We have developed novel genetic systems specifically designed to study NHEJ in budding yeast. Their essential features are creation of a chromosome break by expressed endonucleases in such a way that simple genetic and/or physical analyses can be used to monitor break formation and subsequent repair. This proposal exploits these methodologies to perform a systematic mutational analysis of NHEJ, toward the following specific aims.
In Specific Aim #1, previous work is extended to address the potential involvement of essential and redundant proteins that we hypothesize to have a high likelihood of participating in NHEJ, specifically SMC proteins, chromatin modifying complexes and checkpoint proteins. The remaining aims are a focused analysis of the multifunctional enzyme complexes with known roles in yeast NHEJ: Mrel 1/Rad50/Xrs2, Ku and DNA ligase IV.
Specific Aim #2 explores the structure-function relationships of these proteins by using separation-offunction analysis to gain insight into their discrete contributions to NHEJ.
Specific Aim #3 seeks to explore the sequence of events in NHEJ by using physical analysis in carefully timed break and repair assays.
Specific Aim #4 seeks to identify NHEJ core complex mutants that are specifically deficient in joining incompatible DNA ends, hypothesized to be deficient in recruiting polymerases and nucleases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Special Emphasis Panel (ZRG1-CDF-2 (90))
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Pelroy, Richard
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Liang, Zhuobin; Sunder, Sham; Nallasivam, Sivakumar et al. (2016) Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining. Nucleic Acids Res 44:2769-81
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