DOMAIN ORGANIZATION OF DNA REPAIR IN HUMAN CELLS

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Through nucleotide excision repair (NER), an organism removes the damaged nucleotides e.g. UV-induced cyclobutane pyrimidine dimers from its genome. There are two sub-pathways of NER; global genome repair (GGR) and transcription coupled repair (TCR). There is some evidence that TCR is more efficient and occurs early. Apparently, RNA polymerase stops at the damaged DNA and the transcription blocking lesion from the transcribed strands of the expressed gene is removed. While much is known about the genetic control of GGR and TCR, very little is known about their cellular localization. The investigators will use 5-iododeoxyuridine (IdU) labeling of repair patches in human fibroblasts with deficiencies either in GGR (Xeroderma Pigmentosa-C) or in TCR (Cockayne's syndrome) to determine the focal sites of DNA synthesis. Pulse-labeling experiments will be done to identify and enumerate the early and late foci of repair synthesis. Using different thymidine analogs, the investigators expect to map the early and late domains of repair and to relate these to chromosomal regions of high and low gene density. They will also analyze the relative distribution and co-localization in interphase nuclei of repair foci and proteins diagnostic of GGR and TCR. It is anticipated that these cytological approaches will complement the basic biochemical understanding of NER and provide new insights into the regulation of GGR and TCR in human cells.