Variable termination sites of DNA polymerases encountering a DNA–protein cross-link

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（DNA-protein cross-links, DPCs）是一类重要的DNA损伤，可由内源性交联剂（如甲醛、乙醛）、电离辐射、癌症化疗药物，以及部分酶的无效催化作用诱导产生。由于其结构体积庞大，这类交联会干扰DNA与RNA的合成及DNA修复过程。DPCs具有极强的遗传毒性，细胞应对此类损伤的能力与诸多化疗干预方案密切相关。 然而，由于缺乏便捷的实验模型，DNA聚合酶与DPCs之间的相互作用研究尚不充分。本研究采用硼氢化钠（NaBH₄）诱导大肠杆菌（E. coli）甲酰胺嘧啶-DNA糖苷酶（formamidopyrimidine-DNA glycosylase）与DNA的捕获技术，构建了三类含DPC的DNA聚合酶底物模型：单链模板中的DPC、双链DNA模板链中的DPC，以及双链DNA非模板（置换）链中的DPC。 本研究针对隶属于A、B、X、Y家族的9种DNA聚合酶，探究了其遭遇DPC时的行为特征：包括大肠杆菌DNA聚合酶I的克列诺片段（Klenow fragment）、水生嗜热菌（Thermus aquaticus）DNA聚合酶I、激烈火球菌（Pyrococcus furiosus）DNA聚合酶、嗜热硫化叶菌（Sulfolobus solfataricus）DNA聚合酶IV、人类DNA聚合酶β、κ及λ，以及噬菌体T4与RB69的DNA聚合酶。 尽管所有受试聚合酶均无法完全绕过DPC，但当DPC位于单链模板或置换链中时，B家族DNA聚合酶（T4、RB69）与Y家族DNA聚合酶IV可将引物延伸至交联位点处。在其他情境下，若聚合酶能够置换下游链，则DNA合成会在单链模板或双链DNA中交联位点前4~5个核苷酸处终止。 我们认为，DNA聚合酶在DPC位点处的终止主要源于酶体在抵靠交联蛋白分子时所承受的未缓解构象张力。