Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5′-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5′-ssDNA, ensuring the correct ssDNA bubble size before cleavage.
Bibliographical noteFunding Information:
King Abdullah University of Science and Technology through Competitive Research Award [URF/1/3764-01-01 (CRG7) to S.M.H. and J.A.T.]; NCI [P01 CA092584 to J.A.T., S.T., I.I., in part]; NCI [R35 CA220430 to J.A.T.]; CPRIT [RP180813 to J.A.T.]; NIEHS [R01ES032786 to S.T., I.I.]; Robert A. Welch Chemistry Chair (to J.A.T.); NSF MCB (to I.I.) Funding for open access charges were provided by the King Abdullah University of Science and Technology through URF/1/3764-01-01 (CRG7).
© 2023 The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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