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。该假说基于以下发现：(I)MMR蛋白特异性识别DNA中不匹配的光产物，以及(Ii)在MMR缺陷细胞中紫外线诱变作用增加，假设如下：在半保守DNA复制过程中，MMR系统识别与模板DNA中与紫外光产物相反的碱基，并将其直接切除和重新合成到新生的错误编码链上，同时防止不匹配光产物的核苷酸切除修复(NER)。大多数研究将并行使用高度保守的细菌和保守的细菌和人类的MMR系统，以阐明与人类MMR基因多态相关的机制差异。将测量它们各自的MutL同系物对人(MSH)和细菌(MutS)蛋白与不匹配的环丁烷二聚体(CPD)和合成DNA低聚体中的[6-4]光产物结合亲和力的影响。使用错配修复熟练的人和细菌的无细胞提取物，将比较各自错配的光产物和碱基错配对照在MMR底物质粒中启动切除区域的相对能力。同样的提取物和底物将被用来比较在切除缝隙填充DNA重新合成过程中绕过光产物的效率和准确性。已知的跨损伤合成活性的影响-E.ColiUmuD‘2C蛋白、人DNA聚合酶Delta-on旁路效率和准确性也将被测定。也将测试MMR蛋白与NER蛋白共同作用于错配的光产物底物的能力，从而防止后者对突变的固定。

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.