Hong Yan, PhD
Office Phone: 215-728-2514
A hallmark of cancer cells is their inability to stably maintain genome. This defect accelerates the accumulation of mutations that promote the development of cancer. Paradoxically, it also renders cancer cells more sensitivity than normal cells to the most commonly used chemotherapeutic drugs, which act by damaging DNA. However, the intrinsic genetic diversity of cancer cells also dictates that some cancer cells might be resistant to drugs and eventually lead to recurrence. The research in my laboratory focuses on two major processes that have the most direct impact on genome maintenance: DNA double-strand break (DSB) repair and DNA lesion replication. These two processes have been extensively studied by genetic analysis and enzymatic characterization, but most of the fundamental mechanistic questions remain poorly understood. We take a biochemical approach: biochemical reconstitution of the reactions in cell-free extracts derived from the eggs of the frog Xenopus leavis, dissect the reaction mechanism at the DNA level, identify the critical proteins, and confirm the results in cells.
We have succeeded in reconstituting single-strand annealing (SSA), which along with homologous recombination (HR) constitute the homology-dependent pathways for DSB repair. Using this system, we have found that the Xenopus Werner syndrome protein (xWRN) plays a pivotal role in SSA, providing the first direct evidence to link this protein to a specific DNA repair pathway. Further analysis led us to the discovery of an xWRN-mediated pathway that processes DSBs into 3’ single-strand tails. We found that xWRN, as a RecQ-type DNA helicase, unwinds DNA ends. The 5’ ss-tail is then degraded by a specific nuclease, which we have purified and identified as the Xenopus DNA2 protein (xDNA2), leaving the 3’ ss-tail as the final product of end processing. Because DNA end processing is a key step for not only SSA but also HR, we are conducting a comprehensive study to identify additional proteins in this reaction. In addition, we are using the Xenopus system to determine the mechanistic roles of two well studied but still poorly understood repair proteins, Mre11 and CtIP, in end processing.
In addition to SSA, we have also succeeded in reconstituting the first in vitro system that uses eukaryotic replication forks to replicate a site-specific lesion. Analysis of this system has revealed many novel insights into lesion replication at the nucleotide level resolution. We are currently determining what proteins are important for lesion replication and how they act mechanistically. This should help elucidate the mechanism for how cells deal with damages in the genome.Description of research projects
- Fazlieva R, Spittle CS, Morrissey D, Hayashi H, Yan H, Matsumoto Y. Proofreading exonuclease activity of human DNA polymerase and its effects on lesion-bypass DNA synthesis. Nuclei Acids Res. 2009;37:2854-66. PubMed
- Liao S, Toczylowski T, Yan H. Identification of the Xenopus DNA2 protein as a major nuclease for the 5' -> 3' strand-specific processing of DNA ends. Nucleic Acids Res. 2008;36:6091-100. PubMed
- Liao SR, Matsumoto Y, Yan H. Biochemical Reconstitution of Abasic DNA Lesion Replication in Xenopus Extracts. Nucleic Acids Research. 2007;35:5422-9. PubMed
- Toczylowski T, Yan H. Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein. J Biol Chem. 2006;281:33198-205. PubMed
- Yan H, McCane J, Toczylowski T, Chen C. Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair. J Cell Biol. 2005;171:217-227. PubMed
- Chen CY, Graham J, Yan H. Evidence for a replication function for FFA-1, the Xenopus ortholog of Werner syndrome protein. J. Cell Biol. 2001;152:985-996.