DNA Repair In Cancer And Senescence

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Summary of work: Werner's Syndrome (WS) is a homozygous recessive disease characterized by early onset of many characteristics of normal aging, such as wrinkling of the skin, graying of the hair, cataracts, diabetes, and osteoporosis. The symptoms of WS begin to appear around the age of puberty, and most patients die before age 50. Because of the acceleration of aging in WS, the study of this disease will hopefully shed light on the degenerative processes that occur in normal aging. Cells from WS patients grow more slowly and senescence at an earlier population doubling than age-matched normal cells, possibly because these cells appear to lose the telomeric ends of their chromosomes at an accelerated rate. In general, WS cells have a high level of genomic instability, with increased amounts of DNA deletions, insertions, and rearrangements. These effects could potentially be the result of defects in DNA repair, replication, and/or recombination, although the actual biochemical defect remains unknown. The gene that is defective in WS, the WRN gene, has recently been identified and characterized. We have made purified WRN protein for use in a number of basic and complex biochemical assays. We are using several avenues to identify and characterize the biochemical defect in WS cells. We have shown that there is a transcriptional defect in WS cells, and that this defect also can be seen in cell extracts in vitro. The WRNp has helicase activity and will unwind small and large DNA duplex constructs. It will also unwind unusual DNA structures such as triple helices and DNA forks. We are comparing the Werner helicase activity to that of another helicase, Bloom, which is mutated in Bloom syndrome. Both WRNp and Bloom interact physically and functionally with another protein, replication protein A, which plays major roles in DNA repair and in replication. WRNp does not readily recognize DNA damage and it binds more efficiently to single stranded than double stranded DNA. The WRNp has another enzymatic activity, a 3-5' exonuclease function. We observe that the exonuclease enzyme is blocked by some forms of DNA damage on the substrate DNA, but not by others. The WRN exonuclease interacts both physically and functionally with the Ku heterodimer protein, which is involved in DNA double strand break repair. Recently, we have discovered a number of new functional and physical protein interactions with Werner protein. They include and interaction with Flap-endonuclease 1, which is involved in Base Excision DNA Repair and replication; and p53, involved with apoptosis and signal transduction. WRNp plays and important role in this pathways as we also find interactions with other proteins involved in this process. We have also detected a physical and functional interaction between WRNp and the telomeric binding protein, TRF2, suggesting that at least some of the WRN protein in the nucleus has a function at telomere ends. Our ongoing and future studies will be directed towards elucidation of the causes of the accelerated aging phenotype in WS, with hope that this knowledge can also be applied to our current understanding of both the aging of cells and organisms in general.