<i>Drosophila</i>
            BLM in Double-Strand Break Repair by Synthesis-Dependent Strand Annealing

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-singlestranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS), 2 a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7-9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13-15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18 -20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with . BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yea...

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“…However, genetic studies indicate that BLM may also play a role in promoting HR (51,52). In this study, we present data FIGURE 8.…”

Section: Discussionsupporting

confidence: 60%

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Bugreev

Mazina

Mazin

2009

Journal of Biological Chemistry

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“…Furthermore, BS cells exhibit a hyperrecombination phenotype and greatly enhanced reciprocal exchanges between sister chromatids, as well as chromatid gaps and breaks. These phenotypes are consistent with recent evidence from Drosophila melanogaster that BLM might be required for synthesis-dependent strand annealing (2). WS and RTS are both associated with cancer predisposition, and WS is associated with premature aging (50,57).…”

supporting

confidence: 78%

Multiple Genetic Pathways Involving the Caenorhabditis elegans Bloom's Syndrome Genes him-6, rad-51, and top-3 Are Needed To Maintain Genome Stability in the Germ Line

Wicky

Alpi

Passannante

et al. 2004

Molecular and Cellular Biology

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Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer. We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene. Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis. Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination. In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast. Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a phenotype that is completely suppressed by rad-51. him-6 and top-3 are thus needed to maintain low levels of double-strand breaks in normally proliferating germ cells, and both act in partial redundant pathways downstream of rad-51 to prevent mitotic catastrophy. Finally, we show that topoisomerase III␣ acts independently during a late stage of meiotic recombination.

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“…Flies mutant for DmBlm (Drosophila melanogaster Blm) show elevated mitotic recombination, nondisjunction, and chromosome loss and are nearly sterile (7)(8)(9), recapitulating many of the features of BLM deficiency in humans. Drosophila Blm has been genetically implicated in synthesis-dependent strand annealing and nonhomologous end-joining repair pathways in response to an induced double-strand break (10)(11)(12). These data are not inconsistent with a potential role for Blm in homologous recombination, because SGS1 has also been shown to function outside of homologous recombination in roles that are independent of TOP3 (13).…”

mentioning

confidence: 47%

Topoisomerase IIIα and Bloom’s helicase can resolve a mobile double Holliday junction substrate through convergent branch migration

Plank

Hsieh

2006

Proc. Natl. Acad. Sci. U.S.A.

138

146

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It has long been suspected that a double Holliday junction (dHJ) could be resolved by a topoisomerase partnered with a helicase by convergent branch migration of the HJs. Genetic analysis of yeast TOP3 and SGS1 has lent considerable evidence to the notion that the protein products of these genes are involved in just such a process, although biochemical analysis of the metabolism of a dHJ has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a dHJ substrate that recapitulates many of the features of an endogenous dHJ and represents a much earlier intermediate in the resolution pathway. Here, we show that Drosophila topoisomerase III␣ (Topo III␣) and Blm (a homolog of Sgs1) are capable of resolving this substrate to non-cross-over products and that this activity is stimulated by replication protein A (RPA). We investigated the ability of other Drosophila topoisomerases to perform this reaction in concert with Blm and RPA and discovered that this resolution activity is unique to Topo III␣. Examination of the mechanism of resolution reveals that Topo III␣, Blm, and RPA resolve this substrate by convergent migration of the two HJs toward each other, collapsing the dHJ. This mechanism stands in contrast to classic resolvase activities that use a structure-specific endonuclease to cleave the HJs.recombination ͉ repair ͉ DNA strand exchange ͉ topological constraint T here are primarily two proposed pathways for the processing of the double Holliday junction (dHJ) intermediate of the double-strand break repair model. Structure-specific endonucleases (resolvases) have been identified that can cleave HJs, with the orientation of the cleavage events dictating whether the resolution results in gene conversion events either with (crossovers, COs) or without (non-COs, NCOs) the exchange of sequences flanking the original break site (1-3). This model of resolution is represented by the lower pathway in Fig. 1. In a second potential pathway, the dHJ is resolved by the convergent branch migration of the two HJs by a topoisomerase and helicase (4,5). This mode of resolution would result exclusively in NCO products and is represented by the upper pathway of Fig. 1. Elegant genetic studies in Saccharomyces cerevisiae have pointed toward topoisomerase III (Topo III) and Sgs1, a recQ family helicase, as a topoisomerase͞helicase pair that may play this role in vivo (6).In higher eukaryotes, there are two isoforms of Topo III, ␣ and ␤, and multiple recQ helicases. Of the five recQ helicases in humans, BLM, WRN, and RecQ4 all have been implicated in syndromes involving genetic instability and susceptibility to a wide range of cancers (6). In Drosophila, there are three recQ helicases: Blm, RecQ4, and RecQ5. Flies mutant for DmBlm (Drosophila melanogaster Blm) show elevated mitotic recombination, nondisjunction, and chromosome loss and are nearly sterile (7-9), recapitulating many of the features of BLM deficiency in humans. Drosophila Blm has been genetically implicated in s...

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Drosophila BLM in Double-Strand Break Repair by Synthesis-Dependent Strand Annealing

Cited by 233 publications

References 22 publications

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Multiple Genetic Pathways Involving the Caenorhabditis elegans Bloom's Syndrome Genes him-6, rad-51, and top-3 Are Needed To Maintain Genome Stability in the Germ Line

Topoisomerase IIIα and Bloom’s helicase can resolve a mobile double Holliday junction substrate through convergent branch migration

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