Function of HMCES in abasic site repair and tolerance

PROJECT SUMMARY Thousands of abasic sites form daily in each of our cells. Many types of environmental toxins that cause alkylation or oxidation of DNA bases to form N7-guanine adducts and 8-oxoguanine induce abasic sites. For example, N-nitrosamines that are found in foods, detergents, solvents, plastics, and agricultural chemicals as well as chemicals like carbon tetracholoride, potassium bromate, and chloroform that induce oxidative stress all increase the frequency of abasic sites in DNA. Failures in managing this ubiquitous form of DNA damage can cause a variety of diseases including cancer. The known mechanisms of repair require an intact DNA duplex; however, abasic sites form more readily in single-stranded DNA where they are impediments to replicative polymerases. We recently discovered a new pathway that detects and process abasic sites in single-stranded DNA. This pathway utilizes HMCES (hydroxyl-methyl cytosine embryonic cell specific) to detect and shield these abasic sites from deleterious processing. HMCES contains an evolutionarily conserved domain (SRAP) that binds DNA and forms a covalent crosslink to abasic sites. This DNA-protein crosslink prevents endonucleases from cleaving the single-stranded DNA, thereby preventing double-strand breaks. In this proposal we will further characterize how this pathway acts to maintain genome stability, and more broadly define how abasic sites are tolerated and repaired in the context of DNA replication. We will utilize state of the art biochemical, genetic, and structural approaches in human cells and cell extracts. Completing these studies will provide a mechanistic understanding of how cells cope with a ubiquitous form of DNA damage generated by important environmental genotoxins.