The objective of this proposal is to understand the mechanism for the 5'strand- specific end processing of DNA double-strand breaks (DSBs) in eukaryotes. DSBs are among the most deleterious types of DNA damages. If not properly repaired, DSBs might cause chromosome deletions or translocations, ultimately leading to premature cell death or oncogenic transformation. Accordingly, many cancer-prone disease genes, such as Werner syndrome protein (WRN), Bloom syndrome gene (BLM), BRCA1, and BRCA2, have been implicated in DSB repair. Despite extensive research, many fundamental mechanistic questions about DSB repair are still poorly understood. Of particular importance is the mechanism for the 5'strand-specific end processing that initiates homology-dependent DSB repair. A biochemical approach has been taken to study DSB repair and DNA end processing in Xenopus egg extracts. Single-strand annealing (SSA), one of the homology-dependent DSB repair pathways, has been successfully reconstituted and shown to be dependent on the Xenopus Werner syndrome protein (xWRN). Further analysis has revealed a novel mechanism for end processing. The end is first unwound by a RecQ-type DNA helicase, mainly xWRN, the 5'ss-tail is then degraded by a 5'->3'ss-DNA exonuclease, mainly the Xenopus homologue of DNA2 (xDNA2), and the final product is a 3'ss-tail. Building on these advances, two specific aims are proposed to more comprehensively investigate the mechanism of DNA end processing by characterizing the enzymatic activities of three key end processing proteins and analyzing how their depletions affect end processing in Xenopus egg extracts.
In specific aim I, the Xenopus homologue of EXO1 (xEXO1) will be studied to determine its mechanistic role in end processing. The nuclease activity of xEXO1 will be characterized and its effect on end processing in Xenopus egg extracts will be analyzed to determine if xEXO1 acts on ss-DNA (similarly to xDNA2) or on ds- DNA (distinctively from xDNA2) and if xWRN modulates the xEXO1 pathway.
In specific aim II, the Xenopus homologues of MRE11 (xMRE11) and CtIP1 (xCtIP1) will be studied to determine their mechanistic roles in end processing. In particular, the role of xMRE11's nuclease activity, which has given rise to many conflicting and confusing observations in other systems, will be rigorously dissected. Together, these studies will help us elucidate one of the most fundamental but least understood processes for DSB repair and the function of two clinically important proteins in genome maintenance.
The research on DNA end processing as proposed in this application has two potential applications to the improvement of public health. It is likely that modulating a protein involved in end processing might render cancer cells hyper-sensitive to chemotherapeutic drugs and radiation therapy that act by breaking DNA double-strands. In addition, end processing might be a rate-limiting step for homology-dependent double-strand break repair pathways in human cells and up-regulating this step might improve the efficiency of gene targeting technology.
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