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Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity.

Schwendener S, Raynard S, Paliwal S, Cheng A, Kanagaraj R, Shevelev I, Stark JM, Sung P, Janscak P - J. Biol. Chem. (2010)

Bottom Line: 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.These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cancer Research, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
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.

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Identification of amino acid residues of RECQ5 that are critical for RECQ5-RAD51 complex formation. Single alanine substitutions were created at the charged and aromatic residues in the region of RECQ5 spanning amino acids 654–674 that was found to be essential for interaction with RAD51. A, CBD pull-down assay was performed with the indicated RECQ5 mutants expressed in E. coli as fusion with CBD. Chitin beads coated with wild-type or mutant forms of RECQ5 were incubated with 293T cell extract (600 μg of protein) as described under “Experimental Procedures.” RAD51 binding was detected by Western blotting using anti-RAD51 antibody (bottom panel). RECQ5 proteins were visualized by Ponceau S staining (top panel). B, wild-type and mutant forms of RECQ5 were expressed ectopically in 293T cells as N-terminal fusions with a (His)6-Xpress epitope tag. Extracts from these cells (800 μg of protein) were incubated with Ni-NTA-beads and bound proteins were analyzed by Western blotting using anti-RAD51 and Omni-probe antibodies. The latter antibody recognizes the (His)6-Xpress tag.
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Figure 2: Identification of amino acid residues of RECQ5 that are critical for RECQ5-RAD51 complex formation. Single alanine substitutions were created at the charged and aromatic residues in the region of RECQ5 spanning amino acids 654–674 that was found to be essential for interaction with RAD51. A, CBD pull-down assay was performed with the indicated RECQ5 mutants expressed in E. coli as fusion with CBD. Chitin beads coated with wild-type or mutant forms of RECQ5 were incubated with 293T cell extract (600 μg of protein) as described under “Experimental Procedures.” RAD51 binding was detected by Western blotting using anti-RAD51 antibody (bottom panel). RECQ5 proteins were visualized by Ponceau S staining (top panel). B, wild-type and mutant forms of RECQ5 were expressed ectopically in 293T cells as N-terminal fusions with a (His)6-Xpress epitope tag. Extracts from these cells (800 μg of protein) were incubated with Ni-NTA-beads and bound proteins were analyzed by Western blotting using anti-RAD51 and Omni-probe antibodies. The latter antibody recognizes the (His)6-Xpress tag.

Mentions: In an attempt to identify amino acid residues of RECQ5 that are critical for RAD51 binding, we generated single alanine substitutions at charged and aromatic residues within the region spanning amino acids 654–674 that was found to be required for RECQ5-RAD51 complex formation (Fig. 1C, lane 8). The following residues of RECQ5 were mutated: Arg-654, Phe-659, Phe-666, and Glu-671. The mutant proteins were again expressed in bacteria as fusions with CBD, immobilized on chitin beads and tested for their ability to bind RAD51 from HEK293T cell extract. We found that the alanine substitutions at Arg-654 and Phe-659 of RECQ5 had no effect on its binding to RAD51, while the F666A substitution abolished RAD51 binding by RECQ5 and the E671A substitution reduced it significantly (Fig. 2A).


Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity.

Schwendener S, Raynard S, Paliwal S, Cheng A, Kanagaraj R, Shevelev I, Stark JM, Sung P, Janscak P - J. Biol. Chem. (2010)

Identification of amino acid residues of RECQ5 that are critical for RECQ5-RAD51 complex formation. Single alanine substitutions were created at the charged and aromatic residues in the region of RECQ5 spanning amino acids 654–674 that was found to be essential for interaction with RAD51. A, CBD pull-down assay was performed with the indicated RECQ5 mutants expressed in E. coli as fusion with CBD. Chitin beads coated with wild-type or mutant forms of RECQ5 were incubated with 293T cell extract (600 μg of protein) as described under “Experimental Procedures.” RAD51 binding was detected by Western blotting using anti-RAD51 antibody (bottom panel). RECQ5 proteins were visualized by Ponceau S staining (top panel). B, wild-type and mutant forms of RECQ5 were expressed ectopically in 293T cells as N-terminal fusions with a (His)6-Xpress epitope tag. Extracts from these cells (800 μg of protein) were incubated with Ni-NTA-beads and bound proteins were analyzed by Western blotting using anti-RAD51 and Omni-probe antibodies. The latter antibody recognizes the (His)6-Xpress tag.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871440&req=5

Figure 2: Identification of amino acid residues of RECQ5 that are critical for RECQ5-RAD51 complex formation. Single alanine substitutions were created at the charged and aromatic residues in the region of RECQ5 spanning amino acids 654–674 that was found to be essential for interaction with RAD51. A, CBD pull-down assay was performed with the indicated RECQ5 mutants expressed in E. coli as fusion with CBD. Chitin beads coated with wild-type or mutant forms of RECQ5 were incubated with 293T cell extract (600 μg of protein) as described under “Experimental Procedures.” RAD51 binding was detected by Western blotting using anti-RAD51 antibody (bottom panel). RECQ5 proteins were visualized by Ponceau S staining (top panel). B, wild-type and mutant forms of RECQ5 were expressed ectopically in 293T cells as N-terminal fusions with a (His)6-Xpress epitope tag. Extracts from these cells (800 μg of protein) were incubated with Ni-NTA-beads and bound proteins were analyzed by Western blotting using anti-RAD51 and Omni-probe antibodies. The latter antibody recognizes the (His)6-Xpress tag.
Mentions: In an attempt to identify amino acid residues of RECQ5 that are critical for RAD51 binding, we generated single alanine substitutions at charged and aromatic residues within the region spanning amino acids 654–674 that was found to be required for RECQ5-RAD51 complex formation (Fig. 1C, lane 8). The following residues of RECQ5 were mutated: Arg-654, Phe-659, Phe-666, and Glu-671. The mutant proteins were again expressed in bacteria as fusions with CBD, immobilized on chitin beads and tested for their ability to bind RAD51 from HEK293T cell extract. We found that the alanine substitutions at Arg-654 and Phe-659 of RECQ5 had no effect on its binding to RAD51, while the F666A substitution abolished RAD51 binding by RECQ5 and the E671A substitution reduced it significantly (Fig. 2A).

Bottom Line: 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.These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cancer Research, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
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.

Show MeSH