Human GEN1 and the SLX4-Associated Nucleases MUS81 and SLX1 Are Essential for the Resolution of Replication-Induced Holliday Junctions

Abstract

Summary Holliday junctions (HJs), the DNA intermediates of homologous recombination need to be faithfully processed in order to preserve genome integrity. In human cells the BLM helicase complex promotes non-nucleolytic dissolution of double HJs. In vitro, HJs may be nucleolytically processed by MUS81-EME1, GEN1, and SLX4-SLX1. Here, we exploit human SLX4-null cells to examine the requirements for HJ resolution in vivo. Lack of BLM and SLX4 or GEN1 and SLX4 are synthetically lethal in the absence of exogenous DNA damage with lethality being a consequence of dysfunctional mitosis proceeding in the presence of unprocessed HJs. Thus, GEN1 activity cannot substitute for the SLX4-associated nucleases and one of the HJ resolvase activities, either those associated with SLX4 or GEN1 is required for cell viability even in the presence of BLM. In-vivo HJ resolution depends on both SLX4-associated MUS81-EME1 and SLX1, suggesting that they are acting in concert in the context of SLX4.

Resolution of single and double Holliday junction recombination intermediates by GEN1

“…The activity of Mus81-Mms4, which is low in S phase, is activated by Cdk/Cdc5-mediated phosphorylation events that occur in G2/M phase of the cell cycle (34,35,51,52). The situation in human cells is similar, except that phosphorylation stimulates MUS81-EME1 to interact with the conserved GIY-YIG endonuclease SLX1-SLX4 to form an SLX1-SLX4-MUS81-EME1 (SLX-MUS) complex in prometaphase (49,53,54). The SLX-MUS complex promotes efficient HJ resolution, with SLX1-SLX4 introducing the initial nick at the junction and MUS81-EME1 catalyzing the second incision in the opposing strand (49).…”

Defects in homologous recombination repair behind the human diseases: FA and HBOC

“…This mechanism is termed 'synthesis-dependent strand annealing (SDSA) pathway', and the final product does not contain crossover events (non-crossover). In some cases, the recombination intermediates are converted into a 'double Holliday junction' and subsequently resolved by Holliday junction resolvases (e.g., GEN1 or the SLX4 complex) (Garner et al 2013, Wyatt et al 2013) (with or without crossover) or the BLM helicase complex (without crossover) (Wu & Hickson 2003). For more complete discussion of HR mechanisms such as their relationship with competing NHEJ (the pathway choice) or Holliday junction resolvases, readers should refer to the recent excellent reviews (Chapman et al 2012, Sarbajna & West 2014.…”

Drosophila FANCM Helicase Prevents Spontaneous Mitotic Crossovers Generated by the MUS81 and SLX1 Nucleases

“…In the absence of FANCM, an HJ-containing intermediate is generated, and it is the resolution of this intermediate by MUS81 or SLX1 that generates a crossover. Recent in vitro experiments suggest that vertebrate MUS81 and SLX1 collaborate to resolve HJs, with SLX1 making an initial nick and MUS81 making a second nick and both being coordinated by the MUS312 ortholog SLX4 (Castor et al 2013;Garner et al 2013;Wyatt et al 2013). Our result is more consistent with MUS81 and SLX1 having redundant functions.…”

“…This apparent paradox suggested models in which crossovers are generated by cleavage of a D loop (Whitby 2005) or a structure with nicked HJs (Osman et al 2003;Schwartz and Heyer 2011). Another solution suggested by recent studies is that Mus81-Mms4/Eme1 functions as an HJ resolvase together with the Slx1 nuclease, with Slx1 making the first nick and Mus81 then cutting the nicked HJ (Castor et al 2013;Garner et al 2013;Wyatt et al 2013). Human SLX1 was previously shown to have HJ resolution activity in vitro; this activity is dependent on SLX4, a scaffolding protein that also interacts with MUS81-EME1 (Fekairi et al 2009;Muñoz et al 2009;Svendsen et al 2009).…”