Novel pro- and anti-recombination activities of the Bloom's syndrome helicase

Abstract

Bloom's syndrome (BS) is an autosomal recessive disorder characterized by a strong cancer predisposition. The defining feature of BS is extreme genome instability. The gene mutated in Bloom's syndrome, BLM, encodes a DNA helicase (BLM) of the RecQ family. BLM plays a role in homologous recombination; however, its exact function remains controversial. Mutations in the BLM cause hyperrecombination between sister chromatids and homologous chromosomes, indicating an anti-recombination role. Conversely, other data show that BLM is required for recombination. It was previously shown that in vitro BLM helicase promotes disruption of recombination intermediates, regression of stalled replication forks, and dissolution of double Holliday junctions. Here, we demonstrate two novel activities of BLM: disruption of the Rad51-ssDNA (single-stranded DNA) filament, an active species that promotes homologous recombination, and stimulation of DNA repair synthesis. Using in vitro reconstitution reactions, we analyzed how different biochemical activities of BLM contribute to its functions in homologous recombination.

Figure 1.

BLM inhibits DNA strand exchange activity of hRad51 protein by interaction with the hRad51-ssDNA filament. (A) The experimental scheme. The asterisk indicates the ³²P label. (B) BLM (100 nM) was added to the hRad51 (lanes 1,2) or hDmc1 (lanes 4,5) nucleoprotein filaments assembled on ssDNA in the presence of 1 mM Mg²⁺. (Lane 3) In the control, BLM K695R (100 nM) was added to the hRad51-ssDNA nucleoprotein filament. D-loop formation was initiated by addition of 2 mM CaCl₂ and pUC19 scDNA. (Lane 2) BLM inhibits D-loop formation, if added to the hRad51-ssDNA filament before Ca²⁺. In lanes 6 and 7, BLM (100 nM) was added to the hRad51-ssDNA filament formed in the presence of both 1 mM Mg²⁺ and 2 mM Ca²⁺. D-loop formation was initiated by addition of pUC19 scDNA. D-loops were analyzed by electrophoresis in a 1% agarose gel. (C) The results from B represented as a graph. (D) BLM in indicated concentrations was added to the nucleoprotein filaments formed by either hRad51 (lanes 1–7) or yeast Rad51 (lanes 8–12) proteins in the presence of Mg²⁺. D-loop formation was initiated by addition of either 2 mM CaCl₂ (for hRad51) or 100 nM yeast Rad54 (for yeast Rad51) and pUC19 scDNA. (E) The results from D demonstrating inhibition of hRad51 by BLM are presented as a graph. Error bars in C and D indicate SEM.

Figure 2.

BLM promotes dissociation of the hRad51-ssDNA filament. (A) The experimental scheme of the restriction endonuclease protection assay. The asterisk indicates the ³²P label. (B) The products of DNA cleavage were analyzed in a 10% polyacrylamide gel. BLM was added to the preformed hRad51-ssDNA filament, followed by addition of ³²P-labeled dsDNA containing DdeI cleavage site. (Lanes 6–14) Protection against DdeI cleavage indicates the transfer of hRad51 from the nucleoprotein filament to the dsDNA probe (“hRad51-ssDNA”). In control, hRad51 was replaced by storage buffer (lanes 2–5, “no hRad51”), or reactions were carried out in the presence of AMP-PNP, a nonhydrolyzable ATP analog (lanes 15–17, “hRad51-ssDNA AMP-PNP”). Lane 1 shows migration of original dsDNA fragment noncleaved by DdeI. (C) The results from B presented as a graph. Error bars indicate SEM.

Figure 3.

Analysis of hRad51-ssDNA filament disruption by BLM using electron microscopy. (A) The hRad51-ssDNA filament (indicated by arrows) was formed and then mixed with hRPA. hRad51-ssDNA filament formed in the absence of hRPA is shown in the bottom left corner. (B) hRPA–ssDNA complexes (in the encircled area) are indicated by arrows. (C) The hRad51-ssDNA filament was formed and then mixed with BLM and hRPA. The reaction resulted in disruption of the filament and formation of hRPA–ssDNA complexes (indicated by arrows). Bar, 50 nm.

Figure 4.

ATPase activity of BLM is stimulated by ssDNA stronger than by linear or supercoiled (sc) dsDNA. ATP hydrolysis was carried out in the presence of poly dT (black squares), ϕX174 ssDNA (open squares), pUC19 scDNA (closed circles), or pUC19 linear dsDNA (open circles), or without DNA (diamonds). Error bars indicate SEM.

Figure 5.

BLM promotes disruption of “native” D-loops containing hRad51. (A) The experimental scheme. The asterisk indicates the ³²P label. (B) BLM cannot disrupt “native” D-loops in the presence of Ca²⁺. “Native” D-loops were formed by hRad51, and the reaction was initiated by addition of BLM (lane 2) or BLM storage buffer as a control (lane 1). The DNA products of D-loop disruption were analyzed by electrophoresis in a 1% agarose gel. (C) BLM (lanes 2–5) and hRad54 (lanes 6–9) can disrupt D-loops after Ca²⁺ removal. The protein concentrations are indicated above the gel. In lane 1, proteins were replaced by BLM storage buffer. (D) The data from C presented as a graph. Error bars indicate SEM.

Figure 6.

BLM promotes unwinding of a model replication fork and stimulates primer extension by DNA polymerase η. (A) The experimental scheme. The asterisk indicates the ³²P label. (B) BLM shows a preference in unwinding of the displaced DNA strand versus the primer. The DNA products were analyzed by electrophoresis in a 10% PAGE. The reactions were initiated by addition of BLM (10 nM) in the absence (lane 2) or presence (lane 3) of ATP. In controls, BLM was replaced by hRad54 (50 nM) (lane 4) or BLM storage buffer (lane 1). Migration of DNA markers is shown in lanes 5–7. (C) Schematic representation of replication fork extention by DNA polymerase η. The asterisk indicates the ³²P label. (D) Effect of BLM (lanes 2–7) and hRad54 (lanes 8–15) in indicated concentrations on replication fork extension by DNA polymerase η. Lane 1 shows migration of a nonextended primer. The products of DNA synthesis were analyzed by denaturing PAGE.

Figure 7.

The role of BLM helicase in HR and in stalled replication fork repair. (A) By disruption of the Rad51-filament (top) BLM suppresses HR and promotes alternative repair mechanisms such as “template switching,” which involves replication fork regression (formation of the “chicken foot” structures) (bottom). (B) BLM disrupts Rad51 presynaptic filament, or D-loops, when HR cannot proceed further, preventing formation of potentially toxic intermediates and channeling DSBR into other pathways. (C) In D-loops, BLM stimulates primer extension by DNA polymerase η by unwinding DNA template ahead of the DNA polymerase. (Top) BLM-associated proteins, Topo IIIα and BLAP75, may facilitate this process by removing topological constraints that arise during BLM unwinding. (Bottom) BLM, by promoting D-loop disruption, channels DNA repair process into the SDSA pathway. (D) BLM–Topo IIIα–BLAP75 complex promotes resolution of double Holliday junctions, preventing mitotic crossing over.