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

Abstract

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...

Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

“…6B). This observation is in agreement with the biochemical data indicating that the K695A Blm mutant as well as wild type Blm stimulates Rad51-mediated strand exchange between single-stranded DNA and homologous duplex DNA (52). In summary, Blm may promote HR between diverged sequences through an ATPase activityindependent mechanism.…”

“…In both reactions, although the heterologous sequences may strongly destabilize the D-loop structure, Blm may counteract the negative effect of the heterologous sequences on HR. This idea is supported by the accompanying paper by Bugreev et al (52), in which they show that human Blm enhances D-loop formation by about 4-fold on DNA substrates containing 10% mismatches in vitro. This view is also supported by the several studies in Drosophila.…”

“…6B). This idea is supported by the accompanying paper by Bugreev et al (52), which shows that human Blm alone can enhance Rad51-dependent D-loop formation through an ATPase-independent mechanism in vitro. Moreover, a previous biochemical study showed that DmBlm stimulates strand annealing by an ATPase-independent mechanism (47), raising the possibility that Blm promotes strand annealing and thereby stabilizes formed D-loop.…”

Analysis of the Activities of RAD54, a SWI2/SNF2 Protein, Using a Specific Small-molecule Inhibitor

“…Moreover, the inability of the RAD54 ATPasedefective mutants could be attributed to their excessively stable complexes with dsDNA that disrupt the search for homology by RAD51 rather than to their deficiency in DNA translocation. In addition, several other proteins that stimulate DNA strand exchange of RAD51 either do not have an ATPase-dependent DNA translocation ability, like HOP2-MND1 (30,31) and RAD51AP1 (32,33), or do not require it for RAD51 stimulation, like BLM (34). These data indicate that DNA translocation may not be an essential attribute of RAD51-stimulatory proteins.…”

“…12, 1 M RAD51 was incubated with 3 M 32 P-labeled ssDNA (oligo 90, 90-mer) in reaction buffer containing 25 mM Tris acetate (pH 7.5), 0.5 mM CaCl 2 , 100 g/ml BSA, 2 mM DTT, 1 mM ATP, and the ATP-regenerating system (30 units/ml creatine phosphokinase and 20 mM creatine phosphate) for 15 min at 37°C (34). 100 or 200 nM RAD54 or RAD54 K189R was added, followed by addition of 50 M supercoiled pUC19 DNA to initiate D loop formation.…”