Human SNM1A and XPF–ERCC1 collaborate to initiate DNA interstrand cross-link repair

Abstract

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A-and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

“…Previous genetic and cellular studies revealed that XPF‐ERCC1 collaborates with another repair factor, the 5′‐to‐3′ polarity exonuclease SNM1A, during replication‐coupled ICL repair (Wang et al , 2011). Biochemical analysis of purified human SNM1A demonstrated that it loads onto and digests ICL‐containing DNA substrates from either blunt ends, or, and of importance here, a single nick 5′ (upstream) to the ICL (Sengerova et al , 2012; Allerston et al , 2015).…”

“…It gradually progresses to 1 nt from the ICL (“0” position; step a‐ii), and its arrival at the ICL triggers an XPF‐ERCC1‐RPA‐induced incision six nucleotides 5′ to the junction, in a duplex region (step a‐iii). SNM1A loads from these incisions and digests past the ICL, unhooking the ICL from the DNA duplex, leaving a residual single nucleotide moiety (step a‐iv), which has been demonstrated as the reaction product using mass spectrometry to characterise the reaction products of SNM1A activity in previous work (Wang et al , 2011). This enables translesion synthesis to occur and repair of the broken DNA strand via homologous recombination (step a‐v).…”

“…A strong candidate for such an activity is the SNM1A 5′–3′ ICL repair exonuclease (Wang et al , 2011). SNM1A has been demonstrated to participate in replication‐coupled ICL repair, in the same pathway as XPF‐ERCC1 based on the genetic and cellular phenotypes of cells lacking either or both factors (Wang et al , 2011). Moreover, SNM1A possesses a striking capacity to digest DNA‐containing adducts on the substrate strand, including ICLs (Sengerova et al , 2012; Allerston et al , 2015).…”

“…Moreover, SNM1A possesses a striking capacity to digest DNA‐containing adducts on the substrate strand, including ICLs (Sengerova et al , 2012; Allerston et al , 2015). ICL digestion leaves a residual single‐nucleotide adduct tethered to the opposing strand (Wang et al , 2011). Notably, this intermediate is readily detected in the Xenopus cell‐free replication‐coupled repair system as a substrate for the downstream, TLS‐mediated bypass stage of ICL repair (Raschle et al , 2008).…”

“…Several lines of investigation suggest that XPF‐ERCC1, a structure‐selective heterodimeric endonuclease (Park et al , 1995; Sijbers et al , 1996), is likely to be an essential (although not necessarily sufficient) component of the ICL unhooking apparatus. XPF‐ or ERCC1‐deficient mammalian cells are uniquely hypersensitive to ICL‐inducing agents, compared to cells defective in other factors involved in nucleotide excision repair (NER), and suffer replication fork collapse upon ICL induction (De Silva et al , 2000; Niedernhofer et al , 2004; Wang et al , 2011). Furthermore, the Xenopus cell‐free replication‐coupled repair system demonstrated that ICL unhooking is abolished when XPF is immunodepleted, but not MUS81 or FAN1 (Klein Douwel et al , 2014).…”