The choice of nucleotide inserted opposite abasic sites formed within chromosomal DNA reveals the polymerase activities participating in translesion DNA synthesis.
The choice of nucleotide inserted opposite abasic sites formed within chromosomal DNA reveals the polymerase activities participating in translesion DNA synthesis.
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DOI：
10.1016/j.dnarep.2013.07.008
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发表时间：
2013-11
期刊：
影响因子：
3.8
通讯作者：
Gordenin, Dmitry A.
中科院分区：
文献类型：
--
作者：
Chan, Kin;Resnick, Michael A.;Gordenin, Dmitry A.
基因组DNA中的非碱性位点可以是生物系统（包括人类癌症）中诱变的重要来源。这种诱变需要通过专门的DNA聚合酶绕过脱碱基位点的跨损伤DNA合成（TLS）。TLS蛋白的脱碱基位点旁路特异性已经通过多种手段在体内和体外进行了研究，尽管所得出的结论的一般性还不确定。在这里，我们介绍了一组酵母报告菌株，用于研究染色体DNA内许多随机位置的脱碱基位点旁路的体内特异性。当温度升高到37°C时，这些菌株经历了端粒去帽和切除，暴露了长的3′ ssDNA突出端内的报告基因。表达人APOBEC 3G胞嘧啶脱氨酶以在ssDNA中产生尿嘧啶，其通过尿嘧啶-DNA N-糖基化酶切除。在修复合成过程中，易错TLS绕过了产生的脱碱基位点。由于APOBEC 3G严格的基序特异性和脱碱基位点形成仅限于一条DNA链，该系统提供了有关导致突变的脱碱基位点位置的完整信息。我们概括了先前关于REV 1和REV 3的作用的发现。此外，我们发现序列背景可以强烈影响A或C插入的相对频率。我们还发现，Pol 32，一个非必需的共同亚基的Pols δ和pol的缺失，导致残留的低频C插入依赖于Rev 1催化。我们总结了我们的结果在一个详细的模型TLS组件之间的相互作用，导致容易出错的旁路的无碱基位点。我们的研究结果强调了该系统用于研究基因组DNA内多种类型病变的TLS旁路的实用性。
Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, including human cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic site by specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studied by multiple means in vivo and in vitro, although the generality of the conclusions reached have been uncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity of abasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37°C, these strains underwent telomere uncapping and resection that exposed reporter genes within a long 3′ ssDNA overhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which were excised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resulting abasic sites. Because of APOBEC3G’s strict motif specificity and the restriction of abasic site formation to only one DNA strand, this system provides complete information about the location of abasic sites that led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we found that sequence context can strongly influence the relative frequency of A or C insertion. We also found that deletion of Pol32, a non-essential common subunit of Pols δ and ζ, resulted in residual low-frequency C insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplay between TLS components leading to error-prone bypass of abasic sites. Our results underscore the utility of this system for studying TLS bypass of many types of lesions within genomic DNA.
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影响因子：
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作者：
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通讯作者：
Gordenin DA
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作者：
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