NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro.
NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro.
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DOI：
10.1016/j.dnarep.2017.08.011
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发表时间：
2017-11
期刊：
影响因子：
3.8
通讯作者：
Sugiyama T
中科院分区：
文献类型：
--
作者：
Chen Y;Sugiyama T
翻译合成（TLS）是DNA聚合酶（TLS聚合酶）以高错误率绕过未修复的模板损伤的机制。DNA聚合酶η和λ（Polη和Pol λ）是从酵母到人类保守的主要TLS聚合酶。在这项研究中，我们定量的频率碱基取代酵母Polη和Pol上的未损坏的和脱碱基的模板在体外。为了准确定量，我们使用了基于下一代测序（NGS）的方法，其中通过平行测序直接分析DNA产物。在未损伤的模板上，Polη和Pol具有不同的碱基替换模式，模板序列对替换频率的影响也不同。碱基替换频率主要受替换位点上游和下游相邻碱基的影响。因此，我们提出了这些聚合酶在三个碱基的格式的碱基取代签名。在含有脱碱基位点的模板上，Polη在超过50%的TLS产物中在损伤处产生缺失，但是缺失的形成被Pol η的存在抑制。Pol η和Pol n2种聚合酶在体外协同促进了脱碱基位点上的TLS反应，表明这2种聚合酶可以协同进行高效、高保真的TLS。
Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of basesubstitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct basesubstitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.
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