Laboratory of Genome and Protein Engineering, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia.
PLoS One. 2018 Jun 1;13(6):e0198480. doi: 10.1371/journal.pone.0198480. eCollection 2018.
DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.
DNA-蛋白质交联物（DPCs）是由内源性交联剂（如甲醛或乙醛）以及电离辐射、癌症化疗药物和一些酶的失活作用诱导的重要 DNA 损伤。由于其非常庞大的性质，它们预计会干扰 DNA 和 RNA 合成以及 DNA 修复。DPCs 具有高度遗传毒性，细胞处理它们的能力与许多化疗干预措施有关。然而，由于缺乏方便的实验模型，DNA 聚合酶与 DPCs 的相互作用研究甚少。我们使用 NaBH4 诱导的 E. coli (formamidopyrimidine-DNA glycosylase)与 DNA 结合，构建了含有 DPC 的单链模板、双链 DNA 的模板链或双链 DNA 的非模板（置换）链中的模型 DNA 聚合酶底物。研究了属于 A、B、X 和 Y 家族的 9 种 DNA 聚合酶在遇到 DPC 时的行为：E. coli DNA 聚合酶 I 的 Klenow 片段、Thermus aquaticus DNA 聚合酶 I、Pyrococcus furiosus DNA 聚合酶、Sulfolobus solfataricus DNA 聚合酶 IV、人 DNA 聚合酶 β、κ 和 λ，以及噬菌体 T4 和 RB69 的 DNA 聚合酶。尽管在任何情况下都没有一种聚合酶能够完全绕过 DPC，但 B 家族 DNA 聚合酶（T4、RB69）和 Y 家族 DNA 聚合酶 IV 如果 DPC 位于单链模板或置换链中，则能够在交联位点延伸引物。在其他情况下，如果聚合酶能够置换下游链，则在单链模板或双链 DNA 中的交联位点前 4-5 个核苷酸处，DNA 合成停止。我们认为，DNA 聚合酶在 DPC 上的终止主要是由于酶在压过交联蛋白分子时所经历的未缓解的构象应变。
