Mechanism of Replication-Coupled DNA Interstrand Crosslink Repair

Abstract

Summary DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a novel cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the cross-link. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymeras ζ Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Repair in this system is accompanied by activation of the Fanconi anemia and ATR checkpoint pathways. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.

Recruitment and positioning determine the specific role of the XPF ‐ ERCC 1 endonuclease in interstrand crosslink repair

“…Replication intermediates were isolated and digested with HincII, or HincII and SapI, and separated on agarose gel. Repair efficiency, represented by SapI regeneration, was calculated as described (Räschle et al , 2008) and plotted (right panel).…”

“…Preparation of plasmid with a site‐specific cisplatin ICL (pICL), and ICL repair assays were performed as described (Räschle et al , 2008; Enoiu et al , 2012). Briefly pICL was incubated with HSS for 20 min, following addition of two volumes of NPE ( t  = 0) containing 32 P‐α‐dCTP.…”

“…Mutations in any of the 21 currently known FA genes give rise to Fanconi anemia (FA), a cancer susceptibility disorder characterized by cellular sensitivity to ICL‐inducing agents (Kottemann & Smogorzewska, 2013; Dong et al , 2015). Using a Xenopus egg extract‐based assay, we and others have recently elucidated a molecular mechanism of replication‐coupled ICL repair (Fig EV1; Räschle et al , 2008). This mechanism requires the convergence of two replication forks at an ICL during DNA replication (Zhang et al , 2015).…”

“…This mechanism requires the convergence of two replication forks at an ICL during DNA replication (Zhang et al , 2015). Both forks initially stall 20–40 nucleotides from the crosslink followed by CMG helicase unloading allowing one fork to approach to within 1 nucleotide of the crosslink (Räschle et al , 2008; Fu et al , 2011; Long et al , 2011). Dual incisions on either side of the ICL then unhook the lesion from one of the strands.…”

“…This critical repair step requires the endonuclease XPF (FANCQ)‐ERCC1, which is recruited to the ICL by the large scaffold protein SLX4 (FANCP), and depends on the activation of the Fanconi anemia pathway by ubiquitylation of the FANCI‐FANCD2 complex (Knipscheer et al , 2009; Klein Douwel et al , 2014). After unhooking, a nucleotide is inserted across from the adducted base, followed by strand extension by REV1 and polymerase ζ, consisting of REV7 (FANCV) and REV3 (Räschle et al , 2008; Budzowska et al , 2015; Mamrak et al , 2016). This strand now acts as a template for repair of the opposite strand by HR (Long et al , 2011), leading to fully repaired products.…”