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.
Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.DNA repair | genetic instability | chromosomal aberrations D NA double-strand breaks (DSBs) are potentially lethal DNA lesions which may arise spontaneously during DNA replication or result from exposure to ionizing radiation or other DNAdamaging agents (1). To repair DSBs, eukaryotes have developed two DSB repair pathways: nonhomologous end joining and homologous recombination (HR) (2). HR is required for the repair of complex double-strand lesions such as crosslinks or one-ended DSBs that occur with a cleaved replication fork; nonhomologous end joining appears to have little role in the repair of these lesions. The absence of Rad51 in proliferating cells (and therefore any measurable HR) results in cell lethality. The loss of function of proteins involved in HR, such as breast cancer 2, early onset (BRCA2), will be viable only if there is a BRCA2-independent pathway for Rad51 function.In Saccharomyces cerevisiae, the Rad52 protein plays a key role in HR (3). However, in vertebrates, knockouts of the Rad52 gene show little phenotype, with no obvious defect in HR. Rad52 knockout mice exhibit a nearly normal phenotype, and Rad52-deficient embryonic stem cells are not hypersensitive to agents that induce DSBs, either simple or complex (4, 5). In contrast, Rad51 knockout is embryonically lethal (6, 7), and depletion of Rad51 from vertebrate cells results in an accumulation of chromosome aberrations and subsequent cell death (8). These findings indicate the essential role of Rad51 in the maintenance of chromosomal DNA during the mitotic cell cycle, but the role for Rad52 in vertebrate cells is unclear.Accumulating evidence implicates BRCA2 as an integral component of the HR machinery via the direct regulation of the assembly of Rad51 filaments and its subsequent activity in strand exchange (9-11). Biochemical studies showed that the Ustilago maydis BRCA2 ortholog, Brh2, is involved in the recruitment of Rad51 to the sites of HR; Rad51 then mediates the displacement of replication protein A...
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.
Bloom syndrome (BS), an autosomal recessive disorder, is marked by a high incidence of cancer early in life. Cells derived from BS patients are unstable genetically and exhibit frequent sister chromatid exchanges, reflective of homologous recombination (HR) deregulation. BLM, the RecQ-like helicase mutated in BS, is found in several cellular protein complexes, all of which contain topoisomerase III␣ (Topo III␣) and a novel protein BLAP75. Here, using highly purified human proteins, we show that BLAP75 associates independently with both Topo III␣ and BLM. Even though BLM and Topo III␣ can dissolve the double Holliday junction (DHJ) to yield non-crossover recombinants (1), under physiological conditions, DHJ dissolution becomes completely dependent on BLAP75. The effect of BLAP75 on BLM-Topo III␣ is highly specific, as it is not seen with the combination of Topo III␣ and Escherichia coli RecQ helicase or another human RecQ-like helicase WRN. Thus, BLM, Topo III␣, and BLAP75 constitute a dissolvasome complex that processes HR intermediates to limit DNA crossover formation. This function of the BLM-Topo III␣-BLAP75 dissolvasome is likely indispensable for genome maintenance and cancer avoidance. Cells from Bloom syndrome (BS)3 patients exhibit highly elevated levels of sister chromatid exchanges, indicative of an impairment of the ability to regulate crossover recombination. Consistent with this characteristic, BLM, the RecQ-like helicase mutated in BS, has been found to cooperate with the type IA topoisomerase Topo III␣ to resolve the homologous recombination (HR) intermediate that harbors a double Holliday junction (DHJ) into non-crossover recombinants. This DHJ dissolution activity of the BLM-Topo III␣ complex is thought to be critical for the suppression of DNA crossover formation in mitotic cells and cancer avoidance in humans (1).BLAP75 (BLM-Associated Polypeptide, 75 kDa) was first identified by Meetei et al. (2) as a component of several BLM-containing complexes immunoprecipitable from HeLa nuclear extracts. In a subsequent study, it was shown that small interfering RNA-mediated knockdown of BLAP75 causes a decrease in BLM and Topo III␣ protein levels in cells (3). Importantly, BLAP75 depletion phenocopies the increased frequency of sister chromatid exchanges characteristic of BLM-deficient cells (3). Taken together, the available evidence indicates that BLAP75 exists as a complex with BLM and Topo III␣ (henceforth referred to as the BTB complex), but the mechanistic details of this relationship remain elusive. For instance, whether BLAP75 associates directly with BLM, or through Topo III␣, is unknown. More importantly, it is not clear whether BLAP75 influences the DHJ dissolution activity of BLM-Topo III␣ or, as suggested previously (3), serves as a structural component to promote protein complex formation. In this study, we have carried out biochemical analyses to define the role of the BTB complex in DHJ dissolution. EXPERIMENTAL PROCEDURESExpression and Purification of the BLAP75 Protein-BLAP75 cDNA (fro...
Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and Rad51C, are associated in a stable complex. Rad51B-Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities. We also examined the functional interaction of Rad51B-Rad51C with Rad51 and RPA. Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency. The competition by RPA for substrate binding can be partially alleviated by Rad51B-Rad51C. This recombination mediator function of Rad51B-Rad51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo. Studies in Saccharomyces cerevisiae have identified a large number of genetic loci required for mitotic and meiotic recombination. These genes, comprising RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11, and XRS2 are collectively known as the RAD52 epistasis group. The RAD52 group of genes are also intimately involved in the repair of DNA double-strand breaks induced by exogenous agents such as ionizing radiation (Paques and Haber 1999;Sung et al. 2000) and for telomere maintenance in the absence of telomerase.Cloning, genetic, and biochemical studies have indicated that the structure and function of the RAD52 group genes are highly conserved among eukaryotes, from yeast to humans Thompson and Schild 2001). Interestingly, in mammals, the efficiency of recombination and DNA double-strand break repair is contingent upon the integrity of the tumor suppressors BRCA1 and BRCA2 (Dasika et al. 1999;Moynahan et al. 1999Moynahan et al. , 2001Thompson and Schild 2001), underscoring the importance for deciphering the mechanistic basis of the recombination machinery.In recombination processes that involve the formation of a DNA double-strand break, the ends of the DNA break are processed to yield single-stranded DNA tails. These DNA tails are utilized by the RAD52 group recombination factors for the formation of DNA joints with a homologous DNA template, contained within the sister chromatid or the chromosomal homolog. The nascent DNA joints are then extended in length by branch migration, followed by resolution of DNA intermediates to complete the recombination process (Paques and Haber 1999;Sung et al. 2000).The RAD51 encoded product is the functional homolog of Escherichia coli RecA protein, and like RecA, possesses the ability to promote the homologous DNA pairing and strand exchange reaction that forms heteroduplex DNA joints. In mediating homologous DNA pairing and strand exchange, Rad51 must first assemble onto ssDNA as a nucleoprotein filament, in which the DNA is held in a highly extended conformation (Ogawa et al. 1993;Benson et al. 1994;Sung and Robberson 1995). Assembly of the Rad51-ssDNA nucleoprotein filament is rate-limiting and strongly inhibited by secondary structure in the ssDNA template. The removal of seconda...
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