Distinct roles of XPF-ERCC1 and Rad1-Rad10-Saw1 in replication-coupled and uncoupled inter-strand crosslink repair

Ja-Hwan Seol,Xiaolei Li,Cory Holland,Ilya J Finkelstein,Eun Yong Shim,Sang Eun Lee,Ignacio F Gallardo,Christopher Kim,Robin Eichmiller,Fuyang Li,Melisa Medina-Rivera,Jennifer A Surtees,Paul Hasty

doi:10.1038/s41467-018-04327-0

Abstract

Yeast Rad1–Rad10 (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1–Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1–Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1–Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1–Rad10 removes ICL via NER, whereas in S/G2, Rad1–Rad10 facilitates NER-independent replication-coupled ICLR.

Highlights

Yeast Rad1–Rad[10] (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agentinduced DNA lesions, and contributes to multiple DNA repair pathways
To assess inter-strand crosslink repair (ICLR) at different cell cycle stages, we arrested cells in G1 with α-factor and either immediately exposed them to the HN2 cross-linking agent or released them into S phase before exposure to the drug
The HN2 sensitivity of saw1Δ mus81Δ cells were comparable to that of rad52Δ cells (Fig. 1a). Combined these results suggest that Rad1–Rad10–Saw[1] and Mus81-Mms[4] contribute to most ICLR during S/G2 during which homologous recombination (HR) is critical

Summary

Introduction

Yeast Rad1–Rad[10] (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agentinduced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad[10] catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1–Rad[10] and bind stalled replication forks. Bi-functional alkylating compounds covalently link the two strands of the DNA double helix together, forming interstrand crosslink lesions (ICLs), preventing the separation of the two strands and interfering with essential DNA transactions[1]. As a result, these compounds preferentially kill proliferating cells and have been widely administered as primary chemotherapeutic treatments for numerous types of cancers[1]. Rad[14] (the yeast XPA ortholog) recruits Rad1–Rad[10] to NER substrates[25,26,27], whereas Saw[1] directs Rad1–Rad[10] to 3′ flaps by physical interaction in HR28

Methods

Results

Conclusion