Cranford Matthew T, Dahmen Steven N, Cortez David, Dewar James M
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
Nucleic Acids Res. 2025 Sep 23;53(18). doi: 10.1093/nar/gkaf975.
Abasic sites are frequent DNA lesions that interfere with replication and exert complex biological effects because they can be processed into other lesions. Thus, it remains poorly understood how abasic sites affect replisome progression, which repair pathways they elicit, and whether this depends on the template strand damaged. Using Xenopus egg extracts, we developed an approach to analyze replication of DNA containing a site-specific, stable abasic site on the leading or lagging strand template. We show that abasic sites robustly stall DNA synthesis but exert strand-specific effects. Leading strand abasic sites stall leading strands at the lesion, while lagging strands stall downstream at template-dependent positions. We conclude that replisomes uncouple at leading strand lesions, then stall due to additional template constraints. Synthesis restarts upon lesion bypass or when a converging fork triggers termination. In contrast, lagging strand abasic sites stall only lagging strands, indicating replisome progression was unaffected. Lagging strands reprime downstream, generating a post-replicative gap that is subsequently filled. Despite different effects on replisome progression, both leading and lagging strand abasic sites require translesion DNA synthesis for bypass. Our results reveal how strand-specific abasic sites differentially affect replication and demonstrate that uncoupled replisomes are susceptible to downstream template constraints.
无碱基位点是常见的DNA损伤，会干扰复制并产生复杂的生物学效应，因为它们可被加工成其他损伤。因此，目前对于无碱基位点如何影响复制体进程、引发何种修复途径以及这是否取决于受损的模板链，人们仍知之甚少。利用非洲爪蟾卵提取物，我们开发了一种方法来分析在领先链或滞后链模板上含有位点特异性、稳定无碱基位点的DNA的复制情况。我们发现，无碱基位点会有力地阻碍DNA合成，但会产生链特异性效应。领先链上的无碱基位点会在损伤处使领先链停滞，而滞后链则会在下游模板依赖位置停滞。我们得出结论，复制体在领先链损伤处解偶联，然后由于额外的模板限制而停滞。损伤跨越或汇聚叉触发终止时，合成重新启动。相比之下，滞后链上的无碱基位点仅使滞后链停滞，表明复制体进程未受影响。滞后链在下游重新起始，产生一个复制后缺口，随后被填补。尽管对复制体进程有不同影响，但领先链和滞后链上的无碱基位点都需要跨损伤DNA合成来跨越。我们的结果揭示了链特异性无碱基位点如何不同地影响复制，并表明解偶联的复制体易受下游模板限制的影响。
