Structure-function Studies Of Dna Polymerases

工作概述：我们正在研究DNA聚合酶的结构与其功能之间的关系，包括保真度。今年的成就包括以下方面。我们提供了令人信服的证据表明，进行跨损伤DNA合成（TLS）的Y家族DNA聚合酶的保真度非常低。这表明对正确的碱基配对几何形状的放宽要求，并意味着可以严格控制跨损伤合成聚合酶的功能，以防止人类细胞中潜在的致突变DNA合成。在支持这些想法，X射线晶体结构的两个translesion合成聚合酶揭示了一个开放的活性位点，可能施加更少的几何选择性，正确的碱基配对比所有其他家庭的DNA聚合酶。我们提供的证据表明，TLS聚合酶活性位点的核苷酸错位可以产生移码突变。我们发现X家族酶DNA聚合酶λ具有5 '-dR酶活性和几种其他性质，表明其参与由环境胁迫引起的DNA损伤的碱基切除修复。我们提出并测试了一个模型的聚合酶活性位点与DNA小沟的改变的相互作用，解释了不寻常的碱基替换错误的特异性的家庭A DNA聚合酶与氨基酸替换的结合口袋中的新生碱基对。

Summary of Work: We are examining the relationship between the structures of DNA polymerases and their functions, including fidelity. Accomplishments this year include the following. We provided compelling evidence that the fidelity of Family Y DNA polymerases that perform translesion DNA synthesis (TLS) is very low. This suggests a relaxed requirement for correct base pairing geometry and implies that the functions of translesion synthesis polymerases may be tightly controlled to prevent potentially mutagenic DNA synthesis in human cells. In support of these ideas, the X ray crystal structures of two of the translesion synthesis polymerases reveals an open active site that likely imposes less geometric selectivity for correct base pairing than DNA polymerases in all other families. We provided evidence that nucleotide misalignment in the active sites of TLS polymerases can generate frameshift mutations. We discovered that the Family X enzyme DNA polymerase lambda has 5'-dRPase activity and several other properties suggesting its participation in base excsion repair of DNA damage resulting from environmental stress. We proposed and tested a model for altered interactions of the polymerases active site with the DNA minor groove that explains the unusual base substitution error specificity of a Family A DNA polymerase with an amino acid replacement in the binding pocket for the nascent base pair.