The general goal of this project is to increase our understanding of how genomic stability relates to aging and development. This proposal focuses mainly on the roles played by BLM helicase and WRN helicase in a variety of DNA transactions. BLM and WRN are members of the RecQ family of DNA helicases. Defects in BLM and WRN lead to Bloom Syndrome (BS) and Werner Syndrome (WS) respectively. BS is associated with growth deficiency, some features of premature aging, and a strong predisposition to a broad spectrum of cancers. WS is associated with many features of premature aging and a predisposition to cancer. Both disorders are associated with genomic instability. Because of the profound effect that a single mutation in either BLM or WRN has on aging and development in humans, it is of considerable interest to understand the functions of these two helicases. Studies have suggested roles for BLM and WRN in suppression of recombination, suppression of rearrangements between mismatched sequences, and regulation of error-prone double-strand break (DSB) repair. Perhaps surprisingly, most functions assigned to BLM and WRN are based on experiments using extrachromosomal plasmids or cell-free extracts. We propose to exploit an experimental system to investigate the role of BLM and WRN in DNA transactions as they actually occur in human chromosomes. We have engineered DNA constructs containing a mutated selectable marker gene and a closely linked sequence that can restore function to the marker through recombination. Constructs are stably transfected into cell lines of interest. A DSB can be introduced into the marker gene by transient expression of endonuclease I-SceI and so we can study spontaneous or DSB-induced recombination as well as other DSB repair events that restore marker function. By comparing features of recombination and DSB repair in BS, WS and normal human cells, we will assess the influence of BLM and WRN on recombination and DSB repair. Additional experiments involving the transfection of BS and WS cells with various mutant alleles of BLM or WRN will help us to further dissect the mechanism(s) by which RecQ helicases participate in DNA transactions and how corruption of helicase function may contribute to normal and abnormal aging and development. Public Health Relevance: Our work will provide detailed information about the roles played by two specific proteins in helping to maintain the integrity of human chromosomes. Loss of either of these proteins is known to be associated with developmental deficiency and premature aging. By enhancing our understanding of the functions of the proteins, we will gain insight into normal and abnormal human aging and development.
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|Moon, Sung-Hwan; Lin, Lin; Zhang, Xinna et al. (2010) Wild-type p53-induced phosphatase 1 dephosphorylates histone variant gamma-H2AX and suppresses DNA double strand break repair. J Biol Chem 285:12935-47|
|Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi et al. (2008) Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase. DNA Repair (Amst) 7:1624-35|