线粒体DNA聚合酶γ介导的跨损伤DNA合成分析
Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.
作者信息
Copeland William C, Kasiviswanathan Rajesh, Longley Matthew J
机构信息
Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Dr, Building 101, Rm E316, Research Triangle Park, NC, 27709, USA.
出版信息
Methods Mol Biol. 2016;1351:19-26. doi: 10.1007/978-1-4939-3040-1_2.
Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.
线粒体DNA由核编码的DNA聚合酶γ（polγ）进行复制，该聚合酶由一个140 kDa的单一催化亚基和一个55 kDa的二聚体辅助亚基组成。线粒体DNA易受多种形式的损伤，包括几种类型的氧化损伤、紫外线诱导的光产物、环境来源的化学加合物，以及化疗药物引起的烷基化和链间交联。尽管这些损伤中的许多会阻断DNA复制，但polγ可以通过在模板损伤对面掺入核苷酸并使DNA引物进一步延伸越过损伤来绕过一些损伤。polγ进行的这种跨损伤合成（TLS）过程可以以无错误或易出错的方式发生。TLS的评估需要通过变性聚丙烯酰胺测序凝胶对寡核苷酸底物和复制产物进行广泛分析。本章介绍了跨损伤DNA合成分析的方案。
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