Baylor University, Department of Chemistry and Biochemistry, One Bear Place, #97348, Waco, TX 76798, USA.
Nucleic Acids Res. 2020 Nov 4;48(19):10986-10997. doi: 10.1093/nar/gkaa803.
During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.
在 DNA 复制过程中，模板链中 8-氧鸟嘌呤（8-oxoG）损伤的存在会导致高保真（HiFi）DNA 聚合酶（Pol）停滞。对 8-oxoG 损伤的早期反应涉及“飞行中”跨损伤合成（TLS），其中招募了专门的 TLS Pol 并取代停滞的 HiFi Pol 进行损伤绕过。TLS 的长度必须足够长以实现有效绕过，但也必须受到调节，以最大程度地减少 TLS Pol 引起的复制错误。TLS Pol 终止和 HiFi Pol 恢复的准确位置（即 TLS 补丁的长度）尚未描述。我们使用稳态和预稳态动力学测定来表征不同古菌聚合酶全酶复合物形成的损伤绕过中间体，这些复合物包括 PCNA123 和 RFC。在 TLS PolY 绕过 8-oxoG 后，产物在模板位置积累在损伤后三个碱基处。PolY 对于随后从 +3 位置延伸到 8-oxoG 之外的延伸效率较差，但这种低效率被 HiFi PolB1 的快速延伸所克服。在该中间体处的 Pol 活性的相互作用表明存在一个明确的位置，TLS Pol 的延伸在此处受到限制，并且在绕过 8-oxoG 后，DNA 底物被交还给 HiFi Pol。
