It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.

中文翻译：

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在人类细胞提取物中结构化DNA模板上进行DNA合成的蛋白质组学分析：NHEJ与复制相关蛋白之间的相互作用。

已经确定，短的反向重复序列会触发人类细胞中的碱基取代诱变。但是，复制机制如何处理结构化DNA尚不清楚。先前已有报道，在无人细胞提取物中，使用结构化单链模板的DNA引物延伸在DNA发夹处被瞬时阻断。在此，报道了与DNA模板结合的蛋白质的蛋白质组学分析，并提供了DNA-PK复合物（DNA-PKcs和Ku异二聚体）识别结构化单链DNA并被其激活的证据。用双链寡核苷酸劫持DNA-PK复合物可导致大量去除停顿位点，并提高DNA延伸效率。相反，DNA-PKcs抑制作用可使其稳定在模板上，以及在非同源末端连接（NHEJ）途径下游起作用的其他蛋白质，尤其是XRCC4-DNA连接酶4复合物和辅因子PAXX。在没有DNA-PKcs活性的情况下，NHEJ因子对DNA的保留与引物延伸的额外停止有关，这表明这些蛋白质阻碍了这些位点的DNA合成进程。总的来说，这些结果提高了可能性，即在叉进行过程中，与形成在ssDNA上的发夹结合后，DNA-PK复合物会干扰体内的复制叉动力学。提示这些蛋白质阻碍了这些位点的DNA合成进程。总的来说，这些结果提高了可能性，即在叉进行过程中，与形成在ssDNA上的发夹结合后，DNA-PK复合物会干扰体内的复制叉动力学。提示这些蛋白质阻碍了这些位点的DNA合成进程。总的来说，这些结果提高了可能性，即在叉进行过程中，与形成在ssDNA上的发夹结合后，DNA-PK复合物会干扰体内的复制叉动力学。