ERCC1\u2013XPF targeting to psoralen\u2013DNA crosslinks depends on XPA and FANCD2

Mariangela Sabatella,Alex Pines,Hannes Lans,Jana Slyskova,Wim Vermeulen

doi:10.1007/s00018-019-03264-5

Abstract

The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1–XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1–XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1–XPF recruitment to ICLs is less well understood. Moreover, specific mutations in XPF have been found to disrupt its function in ICLR but not in NER, but whether this involves differences in lesion targeting is unknown. Here, we imaged GFP-tagged ERCC1, XPF and ICLR-defective XPF mutants to investigate how in human cells ERCC1–XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. Our results confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2. Interestingly, we find that two ICLR-defective XPF mutants (R689S and S786F) are less well recruited to ICLs. These studies highlight the differential mechanisms that regulate ERCC1–XPF activity in DNA repair.

Highlights

To investigate how the activity of ERCC1–XPF in response to interstrand crosslinks (ICLs) is regulated in cells, we studied how the complex is recruited to sites of locally induced psoralen-DNA crosslinks independently of nucleotide excision repair (NER)
We show that in human cells, ERCC1–XPF is involved in the repair of the multiple types of lesions induced by psoralen–DNA crosslinking, as part of the NER machinery via XPA, and as part of replication-dependent interstrand crosslink repair (ICLR) via FANCD2
Our results highlight and help to better understand the multifaceted activity and regulation of ERCC1–XPF in DNA repair, which is essential for a better comprehension of how the multiple DNA repair pathways in which ERCC1–XPF is involved tightly collaborate to protect against cancer and promote health

Summary

Introduction

The heterodimeric ERCC1–XPF complex ( called ERCC1–ERCC4) is a structure-specific endonuclease that plays an essential role in multiple DNA repair pathways by incising DNA repair intermediate fork structures at the junction between single-stranded and double-stranded DNA. ERCC1–XPF excises bulky DNA lesions in nucleotide excision repair (NER) [1], unhooks interstrand crosslinks (ICLs), i.e., covalent linkages between two bases on opposite DNA strands, in interstrand crosslink repair (ICLR) [2,3,4], removes 3′ flaps of DNA intermediate structures in specific double-strand break (DBS) repair pathways [5] and cleaves 3′ overhangs from uncapped telomeres to shorten them [6]. The ERCC1–XPF complex was initially identified as an essential endonuclease in NER [1] This DNA repair pathway removes many different types of DNA lesions that disrupt base-pairing, including photolesions induced by UV light and monoadducts and intrastrand crosslinks induced by

Methods

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Conclusion