Members of the RecQ helicase family play critical roles in genome maintenance. There are five RecQ homologs in mammals, and defects in three of these (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. RECQL and RECQL5 have not been associated with a human disease. Here we show that deletion of Recql5 in mice results in cancer susceptibility. Recql5-deficient cells exhibit elevated frequencies of spontaneous DNA double-strand breaks and homologous recombination (HR) as scored using a reporter that harbors a direct repeat, and are prone to gross chromosomal rearrangements in response to replication stress. To understand how RECQL5 regulates HR, we use purified proteins to demonstrate that human RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. By biochemical means and electron microscopy, we show that RECQL5 displaces Rad51 from single-stranded DNA (ssDNA) in a reaction that requires ATP hydrolysis and RPA. Together, our results identify RECQL5 as an important tumor suppressor that may act by preventing inappropriate HR events via Rad51 presynaptic filament disruption.[Keywords: Recql5 helicase; DNA repair; homologous recombination; tumor suppressor; Rad51 recombinase] Supplemental material is available at http://www.genesdev.org.
Homologous recombination is crucial for the repair of DNA breaks and maintenance of genome stability. In Escherichia coli, homologous recombination is dependent on the RecA protein. In the presence of ATP, RecA mediates the homologous DNA pairing and strand exchange reaction that links recombining DNA molecules. DNA joint formation is initiated through the nucleation of RecA onto single-stranded DNA (ssDNA) to form helical nucleoprotein filaments. Two RecA-like recombinases, Rad51 and Dmc1, exist in eukaryotes. Whereas Rad51 is needed for both mitotic and meiotic recombination events, the function of Dmc1 is restricted to meiosis. Here we examine human Dmc1 protein (hDmc1) for the ability to promote DNA strand exchange, and show that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs. DNA strand exchange requires ATP and is strongly dependent on the heterotrimeric ssDNA-binding molecule replication factor A (RPA). We present evidence that hDmc1-mediated DNA recombination initiates through the nucleation of hDmc1 onto ssDNA to form a helical nucleoprotein filament. The DNA strand exchange activity of hDmc1 is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes.
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
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