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Human DNA helicase B functions in cellular homologous recombination and stimulates Rad51-mediated 5'-3' heteroduplex extension in vitro.

Liu H, Yan P, Fanning E - PLoS ONE (2015)

Bottom Line: In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5'-3' direction.A helicase-defective mutant HDHB failed to promote this reaction.Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5'-3' heteroduplex extension during Rad51-mediated strand exchange in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5'-3' DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5'-3' direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5'-3' heteroduplex extension during Rad51-mediated strand exchange in vitro.

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HDHB depletion impairs RPA late-stage foci formation after IR.(A) U2OS cells transiently expressing GFP-HDHB were stained with different antibodies and observed by immunofluorescence. To visualize ssDNA, cells were grown in BrdU for 24 hours, fixed and stained with anti-BrdU antibody. Left, no IR; Right, cells were fixed at 2 h after 5 Gy IR treatment. (B) Percentage of HDHB-positive cells with Rad51, Rad52 or RPA foci colocalizing with HDHB. 500 cells in three experiments were counted. The mean values ± s.d. are plotted. (C) Example of 5 Gy IR-induced Rad51 and γH2AX foci in HCT116 cells at 1 h or 4 h after irradiation. (D) Percentage of cells with Rad51 foci after IR. Right panel is the mean number of Rad51 foci per cell after IR. Total 1000 cells in three experiments were counted. The mean values ± s.d. are plotted. (E) Percentage of cells with γH2AX foci after IR. (F) Left, example of RPA34 foci in HCT116 cells at 0.5 h, 1 h, 2 h, 4 h after 5 Gy IR. Right, percentage of cells with RPA34 foci after IR. *P<0.05, **P<0.05, ***P<0.05, Student t-test. (G) Left, example of RPA34 foci in U2OS cells transfected with control shRNA or HDHB shRNA at one hour after 5 Gy IR. Right, percentage of cells with RPA34 foci after 5 Gy IR at different time points. *P<0.05, **P<0.05, Student t-test.
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pone.0116852.g003: HDHB depletion impairs RPA late-stage foci formation after IR.(A) U2OS cells transiently expressing GFP-HDHB were stained with different antibodies and observed by immunofluorescence. To visualize ssDNA, cells were grown in BrdU for 24 hours, fixed and stained with anti-BrdU antibody. Left, no IR; Right, cells were fixed at 2 h after 5 Gy IR treatment. (B) Percentage of HDHB-positive cells with Rad51, Rad52 or RPA foci colocalizing with HDHB. 500 cells in three experiments were counted. The mean values ± s.d. are plotted. (C) Example of 5 Gy IR-induced Rad51 and γH2AX foci in HCT116 cells at 1 h or 4 h after irradiation. (D) Percentage of cells with Rad51 foci after IR. Right panel is the mean number of Rad51 foci per cell after IR. Total 1000 cells in three experiments were counted. The mean values ± s.d. are plotted. (E) Percentage of cells with γH2AX foci after IR. (F) Left, example of RPA34 foci in HCT116 cells at 0.5 h, 1 h, 2 h, 4 h after 5 Gy IR. Right, percentage of cells with RPA34 foci after IR. *P<0.05, **P<0.05, ***P<0.05, Student t-test. (G) Left, example of RPA34 foci in U2OS cells transfected with control shRNA or HDHB shRNA at one hour after 5 Gy IR. Right, percentage of cells with RPA34 foci after 5 Gy IR at different time points. *P<0.05, **P<0.05, Student t-test.

Mentions: Rad51 and RPA form foci after ionizing radiation, which co-localize with γH2AX foci, an indicator of double-strand breaks [29], [30]. Ectopically expressed HDHB localizes in nuclear foci induced by DNA damaging agents [14]. To see whether these foci are related to homologous recombination repair, we performed immunofluorescence with anti-Rad51, Rad52, and RPA antibodies in cells. In a significant portion of cells, GFP-HDHB colocalized with ssDNA, RPA, Rad52, and Rad51 in the presence or absence of irradiation (Fig. 3A and 3B).


Human DNA helicase B functions in cellular homologous recombination and stimulates Rad51-mediated 5'-3' heteroduplex extension in vitro.

Liu H, Yan P, Fanning E - PLoS ONE (2015)

HDHB depletion impairs RPA late-stage foci formation after IR.(A) U2OS cells transiently expressing GFP-HDHB were stained with different antibodies and observed by immunofluorescence. To visualize ssDNA, cells were grown in BrdU for 24 hours, fixed and stained with anti-BrdU antibody. Left, no IR; Right, cells were fixed at 2 h after 5 Gy IR treatment. (B) Percentage of HDHB-positive cells with Rad51, Rad52 or RPA foci colocalizing with HDHB. 500 cells in three experiments were counted. The mean values ± s.d. are plotted. (C) Example of 5 Gy IR-induced Rad51 and γH2AX foci in HCT116 cells at 1 h or 4 h after irradiation. (D) Percentage of cells with Rad51 foci after IR. Right panel is the mean number of Rad51 foci per cell after IR. Total 1000 cells in three experiments were counted. The mean values ± s.d. are plotted. (E) Percentage of cells with γH2AX foci after IR. (F) Left, example of RPA34 foci in HCT116 cells at 0.5 h, 1 h, 2 h, 4 h after 5 Gy IR. Right, percentage of cells with RPA34 foci after IR. *P<0.05, **P<0.05, ***P<0.05, Student t-test. (G) Left, example of RPA34 foci in U2OS cells transfected with control shRNA or HDHB shRNA at one hour after 5 Gy IR. Right, percentage of cells with RPA34 foci after 5 Gy IR at different time points. *P<0.05, **P<0.05, Student t-test.
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Related In: Results  -  Collection

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pone.0116852.g003: HDHB depletion impairs RPA late-stage foci formation after IR.(A) U2OS cells transiently expressing GFP-HDHB were stained with different antibodies and observed by immunofluorescence. To visualize ssDNA, cells were grown in BrdU for 24 hours, fixed and stained with anti-BrdU antibody. Left, no IR; Right, cells were fixed at 2 h after 5 Gy IR treatment. (B) Percentage of HDHB-positive cells with Rad51, Rad52 or RPA foci colocalizing with HDHB. 500 cells in three experiments were counted. The mean values ± s.d. are plotted. (C) Example of 5 Gy IR-induced Rad51 and γH2AX foci in HCT116 cells at 1 h or 4 h after irradiation. (D) Percentage of cells with Rad51 foci after IR. Right panel is the mean number of Rad51 foci per cell after IR. Total 1000 cells in three experiments were counted. The mean values ± s.d. are plotted. (E) Percentage of cells with γH2AX foci after IR. (F) Left, example of RPA34 foci in HCT116 cells at 0.5 h, 1 h, 2 h, 4 h after 5 Gy IR. Right, percentage of cells with RPA34 foci after IR. *P<0.05, **P<0.05, ***P<0.05, Student t-test. (G) Left, example of RPA34 foci in U2OS cells transfected with control shRNA or HDHB shRNA at one hour after 5 Gy IR. Right, percentage of cells with RPA34 foci after 5 Gy IR at different time points. *P<0.05, **P<0.05, Student t-test.
Mentions: Rad51 and RPA form foci after ionizing radiation, which co-localize with γH2AX foci, an indicator of double-strand breaks [29], [30]. Ectopically expressed HDHB localizes in nuclear foci induced by DNA damaging agents [14]. To see whether these foci are related to homologous recombination repair, we performed immunofluorescence with anti-Rad51, Rad52, and RPA antibodies in cells. In a significant portion of cells, GFP-HDHB colocalized with ssDNA, RPA, Rad52, and Rad51 in the presence or absence of irradiation (Fig. 3A and 3B).

Bottom Line: In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5'-3' direction.A helicase-defective mutant HDHB failed to promote this reaction.Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5'-3' heteroduplex extension during Rad51-mediated strand exchange in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5'-3' DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5'-3' direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5'-3' heteroduplex extension during Rad51-mediated strand exchange in vitro.

Show MeSH
Related in: MedlinePlus