Abstract
The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.
Highlights
The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR)
The locally refined map shows clear sidechain density throughout with the local resolution ranging from 3.4 Å in the RecA-like domain 1 (RecA1) and RecA-like domain 2 (RecA2) domain cores (Fig. 1e, f) to 7 Å at the periphery of the ERCC1 nuclease-like domain (NLD)
We found that XPF–ERCC1 co-expressed with a truncated form of human SLX4 (XPF–ERCC1–SLX4NTD) showed a six-fold increase in catalytic efficiency (Table 3 and Supplementary Fig. 9a–e)
Summary
The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). XPF–ERCC1 activity is essential for removing helical DNA distortions arising from ultraviolet-induced damage and bulky adducts as part of the nucleotide excision repair (NER) pathway[3]. Mutations in XPF and ERCC1 genes are associated with genetic disorders exhibiting diverse phenotypes These pathologies are caused by defects in the genome maintenance pathways that involve XPF–ERCC1, including xeroderma pigmentosum (XP), Cockayne’s syndrome, Fanconi anaemia (FA), XPFE progeria and cerebro-oculo-facioskeletal syndrome[11,12,13,14,15]. The smaller ERCC1 subunit has no catalytic activity but is structurally related to the XPF CM, consisting of a nuclease-like domain (NLD) and a dsDNAbinding (HhH)[2] domain. We show XPF–ERCC1 adopts an autoinhibited conformer in the absence of DNA in order to prevent promiscuous cleavage and provide structural evidence for the initial steps of XPF–ERCC1 activation upon binding a DNA junction
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