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.

No abstract

No abstract

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

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination.[Keywords: Regulation of meiotic recombination; interhomolog crossovers; Rad51 recombinase; double-strand break repair; homologous recombination] Supplemental material is available at http://www.genesdev.org.

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function. Homologous recombination (HR)3 helps eliminate DNA breaks, cross-links, and other deleterious lesions from chromosomes. A failure in HR leads to cancer formation (1, 2), which aptly underscores the importance of delineating the mechanism of the HR machinery. HR is best understood in the context of DNA double strand break repair wherein the break is processed nucleolytically to yield ssDNA, which serves as the nucleation site for the Rad51 recombinase. Polymerization of Rad51 onto the ssDNA results in a helical protein filament called the presynaptic filament. The presynaptic filament harbors a binding site for duplex DNA and provides the catalytic center for the search of homology in the duplex and the formation of a joint between the ssDNA and duplex substrates. Assembly of the presynaptic filament is slow, rendering it prone to interference by the single strand binding factor RPA. In addition, the timely delivery of Rad51 to the single-stranded recombination substrate is complicated by a high affinity of this recombinase for duplex DNA. In Saccharomyces cerevisiae, several HR factors, termed recombination mediators, act to promote the assembly of the Rad51 presynaptic filament (3, 4).Mutations in BRCA2 account for a significant portion of familial breast and ovarian cancers (5) and can lead to the cancer-prone syndrome Fanconi anemia (2). BRCA2 mutant cells exhibit hypersensitivity to genotoxic agents and HR deficiency (1, 6, 7). BRCA2 harbors eight copies of a Rad51 binding motif called the BRC repeat (8 -10). In addition, the carboxyl terminus of BRCA2 contains a distinct Rad51 binding domain, and phosphorylation of this domain by cyclin-dependent kinases appears to modulate the interaction with Rad51 (11). Crystallographic and biochemical characterization of mouse Brca2 has revealed a DNA binding domain that comprises three oligonucleotide/oligosaccharide binding (OB) folds (12).Several lines of evidence suggest that BRCA2 functions as a recombination mediator. First, as noted above, BRCA2 interacts with Rad51 (10) and possesses ssDNA binding activity (12), characteristics...