The Dissolution of Double Holliday Junctions

Double Holliday junctions (dHJS) are important intermediates of homologous recombination. The separate junctions can each be cleaved by DNA structure-selective endonucleases known as Holliday junction resolvases. Alternatively, double Holliday junctions can be processed by a reaction known as “double Holliday junction dissolution.” This reaction requires the cooperative action of a so-called “dissolvasome” comprising a Holliday junction branch migration enzyme (Sgs1/BLM RecQ helicase) and a type IA topoisomerase (Top3/TopoIIIα) in complex with its OB (oligonucleotide/oligosaccharide binding) fold containing accessory factor (Rmi1). This review details our current knowledge of the dissolution process and the players involved in catalyzing this mechanistically complex means of completing homologous recombination reactions.For decades, homologous recombination (HR) was defined as a mechanism for the production of new allelic combinations during meiosis because it can generate so-called crossing-over (see Mehta and Haber 2014). Crossovers are likely generated by the asymmetric cleavage of a key intermediate in HR, the dHJ, by the action of structure-selective endonucleases called “resolvases” (Fig. 1A) (see Wyatt and West 2014). In addition to its essential function during meiosis, HR has proven to be a crucial DNA repair pathway in mitotic cells. Precisely because it has the potential to generate crossing-over, the resolution of dHJ by resolvases affords a high risk of genomic instability in these circumstances. Indeed, when HR is engaged between two homologous chromosomes or two homeologous sequences, dHJ resolution could lead, respectively, to loss of heterozygosity or gross chromosomal rearrangements. Thus, an alternative mechanism allowing dHJ processing without crossing-over would appear essential when HR is used for DNA repair. Such a mechanism, termed dHJ dissolution, is thought to be a major route for dissipation of dHJs arising from HR repair (LaRocque et al. 2011; Krejci et al. 2012). During dHJ dissolution, the two HJs are branch migrated toward one another until they form a hemicatenated intermediate that can be decatenated by a topoisomerase (Fig. 1B). This sophisticated reaction is performed by the so-called “dissolvasome” complex composed of a specific RecQ helicase (BLM in humans/Sgs1 in budding yeast) and a type IA topoisomerase known as topoisomerase III (Fig. 2; for general reviews about RecQ helicases and topoisomerases, see Champoux 2001; Wang 2002; Bachrati and Hickson 2003; Viard and de la Tour 2007; Chu and Hickson 2009; Vindigni and Hickson 2009.Open in a separate windowFigure 1.Double Holliday junction processing pathways. (A) During HJ resolution, each HJ of a dHJ is cleaved by a structure-selective endonuclease (resolvase). Depending on the combination of cleavage orientations, which can be asymmetric or symmetric, this process can generate both crossover and noncrossover products. In contrast, during dissolution (B), each strand engaged in the dHJ is reassociated with its original complementary strand, preventing exchange of genetic material between the two homologous sequences (and hence generating exclusively noncrossover products). DHJ dissolution (B) is initiated by migration of the HJs toward one another. The fusion/collapse of the two HJs results in a hemicatenated intermediate. Decatenation of this intermediate regenerates the original DNA species present before the initiation of HR.Open in a separate windowFigure 2.Domain organization of RecQ helicases, topoisomerases IA, and RMI proteins. (A) Most of the RecQ helicase members share a superfamily 2 helicase domain (SF2), a RecQ conserved domain (RQC), and a helicase and RNase D carboxy-terminal domain (HRDC). Besides this “RecQ core” domain, some RecQ helicases contain amino-terminal and carboxy-terminal extensions that vary in size, sequence, and functionality (e.g., SLD2 homology domain in RECQ4, and a signature motif in the carboxy-terminal domain of RECQ5). The hatched boxes denote partially degenerate RQC domains. BLM/Sgs1 helicases share a common domain organization, including an amino-terminal extension that includes domains for interaction with both TopoIII/RMI1 (TR) and replication protein A (RPA), in addition to a region that has been proposed to be required for DNA strand exchange (SE) activity. (B) All type IA topoisomerases contain a conserved catalytic domain (topoisomerase IA). Some topoisomerase IA enzymes also exhibit a carboxy-terminal extension, frequently composed of zinc finger motifs (black boxes), which is believed to mediate protein–DNA and protein–protein interactions. The contribution of the carboxy-terminal extension to dissolution is unknown. The regions interacting with other components of the dissolvasome are unknown. (C) In RMI1 proteins, only the DUF1676 and the OB-fold domain 1 (OB1) are conserved from yeast to human. The OB1 associates with both BLM/Sgs1 and topoisomerase III (BT/ST). In addition, human RMI1 exhibits a carboxy-terminal extension, composed of a middle region, which mediates RPA binding, and a second OB fold (OB2), which is able to associate with RMI2. RMI2 is also an OB-fold protein (OB3) that stably associates with the dissolvasome in human cells. In total, therefore, the human RMI1/2 complex contains three OB folds.In this review, we first take a historical look at the experimental evidence that led some groups to formulate the proposal that a reaction akin to dissolution must exist, and which then led Wu and Hickson (2003) to confirm its existence by reconstitution of the dissolution reaction in vitro using purified proteins. Following that, we will review the individual and combined roles of the components of what we will term the dHJ dissolvasome. Although many mechanistic aspects of dHJ dissolution remain obscure, several biochemical studies have provided a general understanding of this conceptually simple, but mechanistically complex, reaction.