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.

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo III␣ and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo III␣-BLAP75/ RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo III␣ and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.[Keywords: RECQ; double Holliday junction; Chromatin; BLAP18/RMI2; BLM] Supplemental material is available at http://www.genesdev.org.

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...

Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) III␣ to form an evolutionarily conserved complex, termed the BTB complex, that functions to regulate homologous recombination. BLAP75 binds DNA, associates with both BLM and Topo III␣, and enhances the ability of the BLM-Topo III␣ pair to branch migrate the Holliday junction (HJ) or dissolve the double Holliday junction (dHJ) structure to yield non-crossover recombinants. Here we seek to understand the relevance of the biochemical attributes of BLAP75 in HJ processing. With the use of a series of BLAP75 protein fragments, we show that the evolutionarily conserved N-terminal third of BLAP75 mediates complex formation with BLM and Topo III␣ and that the DNA binding activity resides in the C-terminal third of this novel protein. Interestingly, the N-terminal third of BLAP75 is just as adept as the full-length protein in the promotion of dHJ dissolution and HJ unwinding by BLMTopo III␣. Thus, the BLAP75 DNA binding activity is dispensable for the ability of the BTB complex to process the HJ in vitro. Lastly, we show that a BLAP75 point mutant (K166A), defective in Topo III␣ interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo III␣. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo III␣.Bloom syndrome is a rare, hereditary disorder characterized by proportional dwarfism, light sensitivity, immunodeficiency, male infertility, and high incidence of various types of cancer (1). Cells derived from patients with Bloom syndrome display a high degree of chromosomal instability, marked by a dramatic increase in the frequency of sister chromatid exchanges that arise from the crossing over of chromatid arms during resolution of homologous recombination (HR) 3 intermediates (2, 3). These results indicate an important function of BLM, the protein mutated in Bloom syndrome, in the suppression of HRmediated crossover events.BLM is one of the five RecQ-like DNA helicases in humans (4, 5). Consistent with its HR regulatory role, BLM is able to dissociate various DNA structures that resemble HR intermediates, such as the D-loop and Holliday junction (HJ) (6 -8). Importantly, BLM cooperates with Topo III␣, a Type 1A topoisomerase, to catalyze the resolution of the double Holliday junction (dHJ) intermediate to generate exclusively non-crossover recombinants, in a process termed "dHJ dissolution" (9). Remarkably, BLM also acts to disrupt the Rad51 presynaptic filament and can stimulate DNA repair synthesis by DNA polymerase (10). All these noted HR-related functions of BLM are strictly dependent on its ATPase activity (9 -11). The ability of BLM to unwind HR intermediates, to mediate the dismantling of the Rad51 presynaptic filament, and to catalyze Topo III␣-dependent dHJ dissolution is likely important for the regulation of HR to limit the formation of crossovers and prevent genome rearrangements induced by crossover HR events (10, 12, 13), whereas the DN...