Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.
Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.
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DOI：
10.1371/journal.pgen.1003149
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发表时间：
2012
期刊：
影响因子：
4.5
通讯作者：
Gordenin DA
中科院分区：
文献类型：
--
作者：
Chan K;Sterling JF;Roberts SA;Bhagwat AS;Resnick MA;Gordenin DA
染色体DNA必须是单链形式，才能进行复制、转录和重组等重要交易。单链DNA(SsDNA)比双链DNA(DsDNA)更容易受到损伤，这是因为氮碱中的化学反应部分暴露得更多。因此，可能存在破坏体内单链DNA区域的试剂，同时对双链DNA保持惰性。为了评估这些制剂带来的潜在危险，我们设计了一种针对芽殖酵母的单链DNA突变报告系统。报告菌株携带cdc13-1温度敏感突变，因此转移到37℃会导致端粒去帽，随后5‘到3’酶切。这暴露了包含三个紧密排列的报告基因的报告区，作为一个长的3‘ssDNA悬垂。我们通过表达修饰的人类单链胞嘧啶脱氨酶APOBEC3G来验证该系统检测单链DNA突变损伤的能力。APOBEC3G诱导了高密度的单链DNA胞嘧啶取代，导致两个报告基因同时频繁失活。然后，我们检查了亚硫酸盐的致突变性，亚硫酸盐是一种活性硫氧化物，人类通过呼吸和食物摄入经常接触到这种氧化物。亚硫酸盐的浓度与一些食品中发现的浓度相似，可以诱导高密度的突变，几乎总是作为单链DNA悬垂链中胞嘧啶的替代，导致至少两个报告基因同时失活。此外，亚硫酸盐在DNA中形成了一种长寿命的加合2‘-脱氧尿嘧啶中间体，该中间体抵抗尿嘧啶-DNA N-糖基酶的切割。在修复合成过程中，这种中间体被容易出错的跨损伤dna合成所绕过，经常涉及Pollζ。我们的结果表明，亚硫酸盐诱导的DNA损伤可能特别有害，因为细胞可能不具备准确修复或绕过此类损伤的手段。细胞的基因组编码在双链DNA中。然而，DNA必须以单链形式短暂存在，才能进行模板转录、复制和修复。由于DNA以单链形式(SsDNA)比双链形式(DsDNA)更容易损伤，因此可以有突变ssDNA的试剂，但不能突变dsDNA。由于现有的实验系统不能识别细胞内特定突变的单链DNA突变剂，我们为此设计了一个酵母菌株系统，包含三个紧密排列的报告基因。我们在报告DNA单链的条件下，将酵母暴露在一种酶(APOBEC3G)或一种化学物质(亚硫酸盐)中，这种化学物质可以修饰胞嘧啶，导致一系列突变，使多个报告基因失活。这两种药物都没有诱导对照菌株的突变，其中报告基因仍然是双链的，证实了两种都是细胞内有效的单链DNA特异性突变剂。最后，我们的方法确定了分子作用机制，通过这些机制，试剂可以在细胞内特异性地突变ssDNA，这一领域值得进行大量研究，因为有报道称，在各种癌症中，ssDNA特异性损伤导致了类似的突变簇，占所有突变的40%。
Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA–specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5′ to 3′ enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3′ ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2′-deoxyuracil intermediate in DNA that was resistant to excision by uracil–DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately. A cell's genome is encoded within double-strand DNA. Yet DNA must exist transiently in single-strand form to template transcription, replication, and repair. As DNA is more prone to damage in single-strand (ssDNA) than double-strand (dsDNA) form, there can be agents that mutate ssDNA, but not dsDNA. Since existing experimental systems cannot identify mutagens specifically mutating ssDNA inside cells, we devised a system of yeast strains, containing three closely-spaced reporter genes, for this purpose. We exposed yeast, under conditions where reporter DNA is single-stranded, either to an enzyme (APOBEC3G) or to a chemical (sulfites) that modifies cytosine, resulting in clusters of mutations that inactivated multiple reporter genes. Neither agent induced mutations in control strains where reporter genes remained double-stranded, confirming that both are potent ssDNA–specific mutagens within cells. Finally, our approach ascertained molecular mechanisms of action by which agents can mutate ssDNA specifically within cells, an area that warrants much investigation following reports that ssDNA–specific damage accounts for similar mutation clusters, and up to 40% of all mutations, in various cancers.
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影响因子：
64.5
作者：
Harris, RS;Bishop, KN;Malim, MH
通讯作者：
Malim, MH
DOI：
10.1038/nrc3185
发表时间：
2012-01-12
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
通讯作者：
--
影响因子：
3.5
作者：
Chen, Kuan-Ming;Martemyanova, Natalia;Harris, Reuben S.
通讯作者：
Harris, Reuben S.
影响因子：
8.3
作者：
JAGIELLO, GM;LIN, JS;DUCAYEN, MB
通讯作者：
DUCAYEN, MB
影响因子：
5.6
作者：
Harjes, Elena;Gross, Phillip J.;Chen, Kuan-Ming;Lu, Yongjian;Shindo, Keisuke;Nowarski, Roni;Gross, John D.;Kotler, Moshe;Harris, Reuben S.;Matsuo, Hiroshi
通讯作者：
Matsuo, Hiroshi