Bipartite stimulatory action of the Hop2–Mnd1 complex on the Rad51 recombinase

Abstract

The HOP2 and MND1 genes are indispensable for meiotic recombination. The products of these genes associate to form a stable heterodimeric complex that binds DNA and stimulates the recombinase activity of Rad51 and Dmc1. Here we conduct molecular studies to delineate the action mechanism of the Hop2-Mnd1 complex. We present evidence to implicate Hop2 as the major DNA-binding subunit and Mnd1 as the prominent Rad51 interaction entity. Hop2-Mnd1 stabilizes the Rad51-single-stranded DNA (ssDNA) nucleoprotein filament, the catalytic intermediate in recombination reactions. We also show that Hop2-Mnd1 enhances the ability of the Rad51-ssDNA nucleoprotein filament to capture duplex DNA, an obligatory step in the formation of the synaptic complex critical for DNA joint formation. Thus, our results unveil a bipartite mechanism of Hop2-Mnd1 in homologous DNA pairing: stabilization of the Rad51 presynaptic filament and duplex DNA capture to enhance synaptic complex formation. HR is best understood in the context of DSB repair. Herein, 3Ј single-stranded DNA (ssDNA) tails derived from the nucleolytic processing of the DSB are engaged by the HR machinery, leading to the targeting and invasion of a homologous chromatid to form a DNA joint called the D-loop. Subsequent steps include DNA synthesis, resolution of DNA intermediates, and ligation (Symington 2002;Sung and Klein 2006). In eukaryotes, the homologous DNA pairing reaction responsible for D-loop formation is catalyzed by one of two recombinase enzymes, Rad51 and Dmc1. Whereas Rad51 is needed for recombination in both mitotic and meiotic cells, the expression of Dmc1 is restricted to meiosis. These two recombinases yield inter-homolog crossovers necessary for bridging the homologous chromosomes to ensure their disjunction in meiosis I (Bishop and Zickler 2004;Neale and Keeney 2006). Both Rad51 and Dmc1 are structurally related to the Escherichia coli recombinase RecA. Like RecA, Rad51 and Dmc1 polymerize on ssDNA in an ATP-dependent manner to form a filamentous structure, referred to as the presynaptic filament. The presynaptic filament engages the duplex DNA partner to yield a ternary complex consisting of the recombinase protein filament, ssDNA, and duplex DNA. DNA homology search leads to the formation of the synaptic complex in which the recombining DNA molecules are aligned in homologous registry, and DNA strand invasion occurs upon the location of a free DNA end in either of the DNA molecules (Radding 1982;Sung and Klein 2006).Genetic studies in Saccharomyces cerevisiae and mice Article is online at http://www.genesdev.org/cgi

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

“…The HOP2-MND1 complex is one such DMC1 partner. Biochemical studies have shown that, like RAD51AP1 (7,8), HOP2-MND1 preferentially binds dsDNA over ssDNA (11,12) and promotes DMC1-mediated D-loop formation via functional synergy with the DMC1 presynaptic filament in duplex capture and synaptic complex assembly (12). We have found no evidence for physical interaction of RAD51AP1 and HOP2-MND1 or functional synergy of these two factors in the enhancement of the DMC1-mediated D-loop reaction.…”

“…We first tested the effect of RAD51AP1 on duplex-DNA capture, using our previously published procedure (11). For this, the DMC1 presynaptic filament was assembled on ssDNA linked to magnetic beads via a streptavidin-biotin bridge.…”

“…The assay followed our published procedure (11). To assemble the presynaptic filament, 4 μL of magnetic beads containing 5′-biotinylated 83-mer dT ssDNA (60 ng, corresponding to 9 μM nucleotides) were incubated with 2.7 μM of DMC1 or SpDmc1 in 18 μL of buffer D for 5 min at 37°C.…”

“…Briefly, the 32 P-labeled 90-mer oligonucleotide substrate (2.4-μM nucleotides) was incubated for 5 min with DMC1 or SpDmc1 (0.8 μM) in 10.5 μL of buffer D (35 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM ATP, 4 mM MgCl 2 , 100 μg∕mL BSA, and 1 mM dithiothreitol), followed by the addition of the indicated amount of RAD51AP1 or ScRad54 in 1 μL and a 5-min incubation. pBluescript replicative form I DNA (35-μM base pairs) was then incorporated in 1 μL, and after a 20-min incubation, the reaction mixtures were analyzed (8,11).…”

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

“…Yeast Rad54 protein, Srs2 helicase, RECQ1 helicase, human Hop2-Mnd1 complex, and yeast and human RPA were purified to near homogeneity from E. coli cells tailored to express them, as described (Sigurdsson et al 2001;Krejci et al 2003;Cui et al 2004;Van Komen et al 2006;Chi et al 2007). The BLM, RECQ1, and WRN helicase preparations have been used in several of our published studies (e.g., Raynard et al 2006;Hu et al 2007) and they possess a level of DNA-dependent ATPase activity as high as or higher than that reported in the literature (Brosh et al 1999(Brosh et al , 2000Cui et al 2004; data not shown) and, as expected (Brosh et al 1999(Brosh et al , 2000Cui et al 2004), are adept at unwinding a HJ test substrate (Supplemental Fig.…”