Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, L606, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA.
Chem Res Toxicol. 2012 Mar 19;25(3):755-62. doi: 10.1021/tx300011w. Epub 2012 Feb 29.
DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N(2)-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N(2)-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3' termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ~500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N(2)-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.
DNA 链间交联 (ICLs) 可以通过需要能够进行跨损伤 DNA 合成 (TLS) 的 DNA 聚合酶的生化途径进行修复。这些途径中 TLS 聚合酶的预期功能是在交联位点的对面和之外插入核苷酸。这些反应的结果可以是无错误的或诱变的。在脱氧鸟嘌呤 (dG) 的环外氮之间形成的 ICL 的 TLS 依赖性修复可导致低频碱基替换，主要是 G 到 T 的颠换。以前，我们在体外证明，人聚合酶 (pol) κ 可以实现对模型丙烯醛介导的 N(2)-dG ICL 的无错误旁路，而 Rev1 可以通过在交联的 dG 对面插入 dC 来促进这种旁路。目前的研究对另外两种人类 DNA 聚合酶，pol η 和 pol ι，就它们对这些损伤的无错误或诱变旁路的潜在贡献进行了特征描述。在单个 dNTP 的存在下，pol η 可以在交联的 dG 对面插入 dA、dG 和 dT，但未明显掺入 dC。仅从 dC 和 dG 3'末端观察到进一步的引物延伸，并且产物的量相对于匹配的未损伤底物较低。在加合物位点之外的旁路产物分析表明，在大多数延伸引物中，dG 存在于交联的 dG 对面，并且经常检测到短的缺失。当 pol ι 被测试其复制该 ICL 的能力时，优先掺入正确的 dC，但未观察到进一步的延伸。在稳态条件下，相对于未损伤的 dG，dC 掺入的效率降低了约 500 倍。因此，除了 pol κ 催化的 N(2)-dG ICL 无错误旁路外，可能还存在另一种替代途径，尽管效率较低。在这条途径中，要么 Rev1 要么 pol ι 可以在损伤部位对面插入 dC，而 pol η 可以进行随后的延伸。
