Misrepair of DNA damage is a hallmark of tumor cells. DNA damage can occur from many endogenous and exogenous sources including environmental toxicants. Alkylation damage caused by a myriad of industrial and consumer based sources is pervasive in the environment. DNA alkylation leads to replication stress and DNA damage. If DNA is alkylated during replication, then the replication fork can collapse and many repair mechanisms can be utilized to repair the damaged DNA. How a cell commits to a specific repair pathway is not completely understood. In budding yeast, the Shu complex is critical in the repair of DNA double-strand breaks (DSB) created by processing of replication forks damaged by DNA alkylating agents. This complex is highly conserved throughout eukaryotes and contains the Rad51 paralogues, proteins that are structurally similar to the central DNA repair protein Rad51. In this study, the investigators aim to elucidate the role of the yeast and human Shu complexes in repair of DNA alkylation damage at a replication fork. They are testing the hypothesis that the Shu complex is a critical key regulator of error-free DNA repair by shuttling DNA intermediates into homologous recombination pathway through its unique protein-protein interactions. Consistent with this hypothesis, mutation of the Shu complex proteins leads to repair by alternative error-prone repair mechanisms. The investigators will test the model that the Shu complex plays an essential role in the DNA repair pathway choice by 1) characterizing the association of the Shu complex with a damaged replication fork and the importance of its protein-protein interactions at the fork and 2) solving the structure of the Shu complex DNA binding subunits while bound to a replication fork-like substrate. These studies will elucidate how the Shu complex interactions with DNA and its protein-protein interactions direct these DNA intermediates into distinct repair mechanisms. Using what is learned in yeast to quickly and efficiently identify key substrates, residues, and protein targets, the investigators will expand their studies into human cell lines where they will investigate the role of the human Shu complex and RAD51 paralogues in repair of alkylation damage caused by environmental toxicants. The investigators will specifically characterize mutations and small nucleotide polymorphisms in the hRAD51 paralogues, which may make these individuals hypersensitive to DNA alkylators and could therefore increase cancer risk. Collectively, these studies will provide key insights into the role of the Shu complex in repair of DNA alkylation damage and elucidate how this complex promotes error-free DNA repair to prevent genetic instability. In addition, the investigators will identify at-risk individuals harboing mutations in these important genes that may be more sensitive to DNA alkylation damage and therefore susceptible to human disease.

Public Health Relevance

Cancer is a leading cause of death in the United States, which can arise, in part, from the accumulation of heritable somatic mutations combined with exposure to environmental toxicants. When DNA is not accurately repaired, genomic instability can result which is a hallmark of tumors. The work described here addresses how the Shu complex repairs DNA damage caused by environmental toxicants and how mis-regulation of this process can lead to increased risk to cancer development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES024872-01
Application #
8812516
Study Section
Special Emphasis Panel (ZES1-JAB-D (1R))
Program Officer
Shaughnessy, Daniel
Project Start
2014-01-01
Project End
2019-10-31
Budget Start
2014-01-01
Budget End
2015-10-31
Support Year
1
Fiscal Year
2015
Total Cost
$396,250
Indirect Cost
$122,123
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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