DNA polymerases η and κ bypass N(2)-guanine-O(6)-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.
DNA polymerases η and κ bypass N(2)-guanine-O(6)-alkylguanine DNA alkyltransferase cross-linked DNA-peptides.
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DOI：
10.1016/j.jbc.2021.101124
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
Guengerich FP
中科院分区：
文献类型：
--
作者：
Ghodke PP;Guengerich FP
当蛋白质在外源性或内源性烷化剂存在下与DNA共价捕获时，形成DNA-蛋白质交联。如果不修复，它们会抑制转录以及复制过程中的DNA解旋，并可能导致基因组不稳定甚至细胞死亡。已知DNA修复蛋白O 6-烷基鸟嘌呤DNA-烷基转移酶（AGT）在致癌物1，2-二溴乙烷存在下形成DNA交联，导致细菌和哺乳动物细胞中的G：C至T：A颠换和其他突变。我们假设AGT-DNA交联将被核蛋白酶处理，产生足够小的肽，以绕过跨损伤（TLS）聚合酶。在这里，来自AGT活性位点的15-mer和36-mer肽通过含有肽脱氢丙氨酸部分的硫醇的缀合物加成交联到鸟嘌呤的N2位置。用DNA聚合酶（pols）η和κ进行的旁路研究表明，两者都可以准确地旁路交联的DNA肽。人（h）pol κ使正确核苷酸dCTP稳态掺入的特异性常数（kcat/Km）增加6倍，hpol η增加3倍，hpol η优先插入核苷酸，顺序为dC > dG > dA > dT。延伸产物的LC-MS/MS分析还揭示了交联的15-mer肽通过hpol η的无错误旁路。我们的结论是，一个庞大的15聚体AGT肽交联的鸟嘌呤的N2位置可以延迟聚合，但整体保真度不受影响，因为只有正确的碱基插入和延伸。
DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.
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