ANTI MUTAGENIC MISMATCH REPAIR OF UV DAMAGED DNA

长期目标是阐明人类和细菌错配修复（MMR）系统对抗紫外线（UV）诱变的机制，并减少紫外线诱导的皮肤癌。基于(i) MMR蛋白特异性识别DNA中不匹配的光产物，而不是“匹配的”光产物，以及（ii）在MMR缺陷细胞中紫外线诱变增加的发现，假设如下：在半保守DNA复制过程中，MMR系统识别模板DNA中不正确的碱基结合的碱基，将相反的UV光产物结合，并将其直接切除和重新合成到新生的错误编码链上，同时防止错配光产物的核苷酸切除修复（NER）。大多数研究将同时使用高度保守的细菌和保守的细菌和人类MMR系统，以阐明与人类MMR多态性相关的机制差异。它们各自的MutL同源物对人（MSH）和细菌（MutS）蛋白对合成DNA低聚物中不匹配的环丁烷二聚体（CPDs）和[6-4]光产物的结合亲和力的影响将被测量。使用精通错配修复的人类和细菌无细胞提取物，将比较各自的错配光产物和碱基错配对照在mmr -底物质粒中启动切除束的相对能力。相同的提取物和底物将用于比较在切除间隙填充DNA再合成过程中绕过光产物的效率和准确性。已知翻译合成活性的影响。大肠杆菌UmuD'2C蛋白，人DNA聚合酶δ on旁路效率和准确性也将被确定。MMR蛋白与NER蛋白在不匹配的光产物底物上完成的能力，从而防止后者的突变固定，也将被测试。

Long-TERM goals are to elucidate mechanisms by which human and bacterial mismatch-repair (MMR) systems antagonize ultraviolet-light (UV) mutagenesis, and reduce UV-induced skin cancer. The hypothesis, based on findings (i) that MMR protein specifically recognize mismatched, but not "matched" photoproducts in DNA, and (ii) that UV mutagenesis is increased in MMR-deficient cells, is as follows: During semi-conservative DNA replication, MMR systems recognize incorrect bases incorporated bases incorporated opposite UV photoproducts in template DNA, and direct excision and resynthesis to the nascent error- encoding strands, while preventing nucleotide excision repair (NER) of the mismatched photoproducts. Most studies will employ the highly conserved bacterial and conserved bacterial and human MMR systems in parallel, to elucidate mechanistic differences relevant to human MMR polymorphisms. Effects of their respective MutL homologs on binding affinities of human (MSH) and bacterial (MutS) proteins for mismatched cyclobutane dimers (CPDs) and [6-4] photoproducts in synthetic DNA oligomers will be measured. Using mismatch-repair-proficient human and bacterial cell-free extracts, the relative abilities of the respective mismatched photoproducts and base-mismatch controls to initiate excision tracts in MMR-substrate plasmids, will be compared. The same extracts and substrates will be used to compare the efficiencies and accuracies with which the photoproducts are bypassed during excision- gap-filling DNA resynthesis. The effects of known translesion-synthesis activities-E. coli UmuD'2C protein, human DNA polymerase delta-on bypass efficiency and accuracy will be determined as well. The abilities of MMR proteins to complete with NER proteins for mismatched- photoproduct substrates, thus preventing mutation-fixation by the latter, will also be tested.