Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta.
Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta.
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DOI：
10.1021/bi800529f
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发表时间：
2008-08-05
期刊：
影响因子：
2.9
通讯作者：
Basu, Ashis K.
中科院分区：
文献类型：
--
作者：
Colis, Laureen C.;Raychaudhury, Paromita;Basu, Ashis K.
本文研究了γ射线或X射线诱发的猴肾细胞（COS-7）和人胚肾细胞（293T）DNA串联损伤G [8，5-Me] T和T [5-Me，8] G链内交联的比较诱变。对于293T细胞中的G [8，5-Me] T，5.8%的后代含有靶向碱基替换，而10.0%显示靶向单碱基替换。在靶向突变中，G → T突变发生频率最高。在交叉连接的2个碱基5 ′和3个碱基3 ′处检测到靶向突变。最常见的靶向突变是C → T转换，紧邻5 ′端的G [8，5-Me] T交联。还检测到移码（4.6%）（主要是小缺失）和多碱基替换（2.7%）。对于T [5-Me，8] G交联，注意到类似的突变模式，但突变频率显著高于G [8，5-Me] T。靶向和非靶向突变的发生频率均为16%，并且均包括显性G → T颠换。与293 T细胞一样，COS细胞中T [5-Me，8] G（11.4%）中发生的靶向突变是G [8，5-Me] T（4.7%）中发生的靶向突变的两倍多。此外，在COS细胞中，相对于G [8，5-Me] T，T [5-Me，8] G中损伤5 ′端的单碱基替换水平增加，损伤3 ′端的单碱基替换水平降低。似乎在交联处或附近的大多数碱基取代是由于与模板碱基相对的dAMP的掺入而导致的，与所谓的"A规则"一致。为了确定人聚合酶η（hpol η）是否可能参与诱变旁路，对相同序列中的G [8，5-Me] T进行了体外旁路研究，结果表明hpol η可以绕过交联，并在每个交联碱基的对面掺入正确的dNMP。对于G [8，5-Me] T，通过hpol η的核苷酸掺入与通过酵母pol η的核苷酸掺入显著不同，因为后者相对于交联的Gua更容易出错。通过hpol η相对的交联T掺入正确的核苷酸dAMP的效率是错误核苷酸的3 - 5倍，而通过交联G相对的dCMP掺入的效率是dTMP的10倍。因此，hpol η的核苷酸掺入模式与观察到的细胞突变不一致。然而，在病变处和病变附近，与对照模板相比，hpol η更容易出错。体外数据表明，translesion合成由另一个Y-家族DNA聚合酶和/或有缺陷的参与的辅助蛋白是一个更有可能的情况下，在哺乳动物细胞中的这些病变的诱变。然而，hpol η可以在正确绕过交联中起作用。
Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links.
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