DNA Repair In Cancer And Senescence

Aging

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Abstract

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 are searching for pathways in which WRN participates and 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,p53, topoisomerase I, telomeric binding factors 1 and 2, c-abl, polymerase beta, and poly (ADP) ribose polymerase. These observations of functional protein interactions corroborated by cell biological observations suggest that WRN protein is involved in DNA repair processes, in partical the pathways of base excision repair and of recombination. We also observe that post translational modifications of the WRN protein such as phosphorylation affect the catalytic properties of the protein and may thus be very important in the regulation of its functions. We have fund that the WRN protein participates in processes at the telomere end and helps unwind the specific DNA substrates that are found there and interacts with proteins localized to those regions. Thus an important function of the WRN protein is to maintain and stabilize the telomere regions and to help repair DNA lesions situated there. 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.