Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA–protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3′-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.

DNA 中的无嘌呤/无嘧啶（AP 或脱碱基）位点是最常见的 DNA 损伤形式之一。AP 位点具有反应性并与蛋白质和 DNA 形成交联，容易发生链断裂，并抑制 DNA 复制和转录。复制相关的 AP 位点修复蛋白 HMCES 保护细胞免于链断裂，抑制诱变跨损伤合成，并通过与 AP 形成稳定的噻唑烷 DNA-蛋白质交联 (DPC) 参与修复源自 AP 位点的链间 DNA 交联单链 DNA (ssDNA) 中的位点。尽管 HMCES 对基因组维持及其催化 SRAP（SOS 响应相关肽酶）结构域的进化保守性很重要，但 DPC 形成和分解的酶促机制尚不清楚。使用细菌同系物 YedK，我们展示了 SRAP 结构域催化 AP 位点转化为其反应性的开环醛形式，并且我们为在更稳定的噻唑烷之前形成的席夫碱中间体提供了结构证据。我们还报告了两项新的活动，其中 SRAP 与双功能 DNA 糖基化酶产生的 DNA 3' 末端的多不饱和醛反应并催化 DPC 的直接逆转以再生 AP 位点，我们在 YedK 和 HMCES-SRAP 中都观察到后者蛋白质。总之，这项工作提供了对 HMCES DPC 在细胞中解决的可能机制的见解。其中 SRAP 与双功能 DNA 糖基化酶产生的 DNA 3' 末端的多不饱和醛反应并催化 DPC 的直接逆转以再生 AP 位点，我们在 YedK 和 HMCES-SRAP 蛋白中都观察到后者。总之，这项工作提供了对 HMCES DPC 在细胞中解决的可能机制的见解。其中 SRAP 与双功能 DNA 糖基化酶产生的 DNA 3' 末端的多不饱和醛反应并催化 DPC 的直接逆转以再生 AP 位点，我们在 YedK 和 HMCES-SRAP 蛋白中都观察到后者。总之，这项工作提供了对 HMCES DPC 在细胞中解决的可能机制的见解。