Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.
Bottom Line: Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool.The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents.Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks.
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.Show MeSH
Related in: MedlinePlus
Mentions: The work presented here shows that SLX4, in complex with XPF-ERCC1, is a far more potent nuclease than XPF-ERCC1 alone. Moreover, SLX4 imparts structural preference on XPF-ERCC1 toward DNA flaps and replication-like structures over stem-loop or bubble substrates (those bearing greater similarity to nucleotide excision repair substrates) without a free 3′ overhang. This stimulation of activity does not appear to be due to enhanced substrate binding, suggesting that its effect is more likely due to altered catalysis on specific substrates. Our data reveal that the individual proteins are present with 2:2:2 stoichiometry in the SXE complex, implying that each SXE complex contains two active sites of XPF. It is tempting to speculate that this may influence enzyme efficiency and potentially provides a mechanism by which enhanced catalysis is achieved. Our studies show that a minimal SXE complex is capable of dual incisions at either side of a DNA crosslink. The SXE complex primarily appears to recognize the 3′ arm of a fork and that cutting occurs 1 and 4 nt from the ss/ds junction. It is well known that XPF-ERCC1 is crucial for crosslink repair and that it can biochemically unhook a crosslink, but it is unclear the efficiency with which it achieves this (Fisher et al., 2008; Kuraoka et al., 2000). Our comparison of SLX4-XPF-ERCC1 complex and XPF-ERCC1 alone shows that SLX4 greatly stimulates this nuclease activity, which can be integrated into a model of ICL repair (Figure 7). Recently, it has been discovered that XPF-ERCC1 is critical for ICL incision in a Xenopus system, dependent upon monoubiquitylated FANCD2 (Klein Douwel et al., 2014; Knipscheer et al., 2009). Furthermore, it was previously shown that the leading strand template remains intact in this process (Räschle et al., 2008). While this may suggest a major difference between the activity we report and what is seen in Xenopus, a few points need to be taken into consideration. The Xenopus model of ICL repair involves two converging replication forks. It is therefore possible that the dual incisions we observe take their cue from the replication fork coming from the opposite direction toward the ICL. This would still leave an intact, adducted parental strand as template for TLS. The in vitro Xenopus system might also have additional factors that specifically restrict the activity of SXE nuclease complex to favor a particular arm (for example, preloading Rad51 onto ssDNA; Long et al., 2011). Indeed, although dependent on monoubiquitylated FANCD2, the mechanism by which SLX4-XPF-ERCC1 is recruited to site of the crosslink is uncertain. The recruitment may occur through the direct interaction of SLX4 and monoubiquitylated FANCD2 or indirectly, through an intermediary (Yamamoto et al., 2011).
Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.