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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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Role of physical interaction between RECQ5 and RAD51 in the suppression of HR-mediated DSB repair in human cells. A, diagram of the DR-GFP reporter along with the homology-directed repair (HDR) product that gives rise to GFP+ cells. B, effect of overexpression of wild-type (WT) RECQ5 and the F666A mutant on HR repair of an I-SceI-induced DSB in HEK293 cells. Cells were transfected with an I-SceI expression vector along with the indicated amounts of either an expression vector for RECQ5 (WT or F666A) or empty vector (EV). Shown are the levels of repair relative to the mean value of a parallel set of EV transfections. Data represent the mean values of three independent transfections. Error bars reflect the S.D. Asterisks denote statistically significant difference as compared with WT RECQ5 by unpaired Student's t test (p = 0.0086 for 0.4 μg of plasmid DNA; p = 0.006 for 0.8 μg of plasmid DNA).
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Figure 5: Role of physical interaction between RECQ5 and RAD51 in the suppression of HR-mediated DSB repair in human cells. A, diagram of the DR-GFP reporter along with the homology-directed repair (HDR) product that gives rise to GFP+ cells. B, effect of overexpression of wild-type (WT) RECQ5 and the F666A mutant on HR repair of an I-SceI-induced DSB in HEK293 cells. Cells were transfected with an I-SceI expression vector along with the indicated amounts of either an expression vector for RECQ5 (WT or F666A) or empty vector (EV). Shown are the levels of repair relative to the mean value of a parallel set of EV transfections. Data represent the mean values of three independent transfections. Error bars reflect the S.D. Asterisks denote statistically significant difference as compared with WT RECQ5 by unpaired Student's t test (p = 0.0086 for 0.4 μg of plasmid DNA; p = 0.006 for 0.8 μg of plasmid DNA).

Mentions: Finally, we sought to investigate the role of the physical interaction between RECQ5 and RAD51 in the anti-recombination activity of RECQ5 in vivo. To this end, we compared the ability for wild-type RECQ5 versus the F666A mutant to exert a trans-dominant negative effect on HR-mediated repair of an endonuclease-generated chromosomal DSB, using the DR-GFP reporter integrated into HEK293 cells (20). In this reporter, the recognition site for the rare cutting endonuclease I-SceI is integrated into a full-length GFP gene (SceGFP) that is followed by an internal GFP fragment (iGFP) (Fig. 5A). Repair of the I-SceI-generated DSB by HR using the iGFP fragment as a template restores a functional GFP gene. Thus, the frequency of HR can be quantified as the percentage of GFP+ cells by FACS analysis. From these experiments, we found that overexpression of wild-type RECQ5 caused a decrease in the frequency of HR (Fig. 5B). The F666A mutant of RECQ5 also reduced the frequency of HR, but this inhibitory effect was significantly lower than that of wild-type RECQ5 (Fig. 5B). These data indicate that the suppression of HR by RECQ5 in vivo is partially dependent on the formation of RECQ5-RAD51 complex.


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)

Role of physical interaction between RECQ5 and RAD51 in the suppression of HR-mediated DSB repair in human cells. A, diagram of the DR-GFP reporter along with the homology-directed repair (HDR) product that gives rise to GFP+ cells. B, effect of overexpression of wild-type (WT) RECQ5 and the F666A mutant on HR repair of an I-SceI-induced DSB in HEK293 cells. Cells were transfected with an I-SceI expression vector along with the indicated amounts of either an expression vector for RECQ5 (WT or F666A) or empty vector (EV). Shown are the levels of repair relative to the mean value of a parallel set of EV transfections. Data represent the mean values of three independent transfections. Error bars reflect the S.D. Asterisks denote statistically significant difference as compared with WT RECQ5 by unpaired Student's t test (p = 0.0086 for 0.4 μg of plasmid DNA; p = 0.006 for 0.8 μg of plasmid DNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Role of physical interaction between RECQ5 and RAD51 in the suppression of HR-mediated DSB repair in human cells. A, diagram of the DR-GFP reporter along with the homology-directed repair (HDR) product that gives rise to GFP+ cells. B, effect of overexpression of wild-type (WT) RECQ5 and the F666A mutant on HR repair of an I-SceI-induced DSB in HEK293 cells. Cells were transfected with an I-SceI expression vector along with the indicated amounts of either an expression vector for RECQ5 (WT or F666A) or empty vector (EV). Shown are the levels of repair relative to the mean value of a parallel set of EV transfections. Data represent the mean values of three independent transfections. Error bars reflect the S.D. Asterisks denote statistically significant difference as compared with WT RECQ5 by unpaired Student's t test (p = 0.0086 for 0.4 μg of plasmid DNA; p = 0.006 for 0.8 μg of plasmid DNA).
Mentions: Finally, we sought to investigate the role of the physical interaction between RECQ5 and RAD51 in the anti-recombination activity of RECQ5 in vivo. To this end, we compared the ability for wild-type RECQ5 versus the F666A mutant to exert a trans-dominant negative effect on HR-mediated repair of an endonuclease-generated chromosomal DSB, using the DR-GFP reporter integrated into HEK293 cells (20). In this reporter, the recognition site for the rare cutting endonuclease I-SceI is integrated into a full-length GFP gene (SceGFP) that is followed by an internal GFP fragment (iGFP) (Fig. 5A). Repair of the I-SceI-generated DSB by HR using the iGFP fragment as a template restores a functional GFP gene. Thus, the frequency of HR can be quantified as the percentage of GFP+ cells by FACS analysis. From these experiments, we found that overexpression of wild-type RECQ5 caused a decrease in the frequency of HR (Fig. 5B). The F666A mutant of RECQ5 also reduced the frequency of HR, but this inhibitory effect was significantly lower than that of wild-type RECQ5 (Fig. 5B). These data indicate that the suppression of HR by RECQ5 in vivo is partially dependent on the formation of RECQ5-RAD51 complex.

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
Related in: MedlinePlus