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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 promotes heteroduplex extension but not joint molecule formation.(A) Strand exchange reaction was performed with 32P-labeled linear dsDNA and in the presence of 50 mM (NH4)2SO4. The concentration of HDHB was 100 nM. (B) Quantitative analysis of jm and nc DNA as the percentage of initial dsDNA. The mean values ± s.d. from three independent experiments are plotted.
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pone.0116852.g005: HDHB promotes heteroduplex extension but not joint molecule formation.(A) Strand exchange reaction was performed with 32P-labeled linear dsDNA and in the presence of 50 mM (NH4)2SO4. The concentration of HDHB was 100 nM. (B) Quantitative analysis of jm and nc DNA as the percentage of initial dsDNA. The mean values ± s.d. from three independent experiments are plotted.

Mentions: To better understand the mechanism of the stimulation, we labeled the 5′-end of the linear dsDNA with 32P and quantified the reaction products. Rad51 promoted the formation of joint molecules gradually in 3 h (Fig. 5). In the presence of HDHB, the formation of nicked circular DNA was significantly stimulated. This was accompanied by the releasing of linear ssDNA (Fig. 5A). The amount of joint molecules and nicked-circular DNA formed in the reactions was quantified. Although more nicked-circular DNA was formed in the presence of HDHB than in its absence, the amount of joint molecules decreased in parallel (Fig. 5B). The total amount of reaction products (joint molecules and nicked circular DNA) was not increased in HDHB-containing reactions as compared to control reaction, suggesting that HDHB stimulates nicked circular DNA formation by promoting the heteroduplex extension, but not the formation of joint molecules.


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 promotes heteroduplex extension but not joint molecule formation.(A) Strand exchange reaction was performed with 32P-labeled linear dsDNA and in the presence of 50 mM (NH4)2SO4. The concentration of HDHB was 100 nM. (B) Quantitative analysis of jm and nc DNA as the percentage of initial dsDNA. The mean values ± s.d. from three independent experiments are plotted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4305318&req=5

pone.0116852.g005: HDHB promotes heteroduplex extension but not joint molecule formation.(A) Strand exchange reaction was performed with 32P-labeled linear dsDNA and in the presence of 50 mM (NH4)2SO4. The concentration of HDHB was 100 nM. (B) Quantitative analysis of jm and nc DNA as the percentage of initial dsDNA. The mean values ± s.d. from three independent experiments are plotted.
Mentions: To better understand the mechanism of the stimulation, we labeled the 5′-end of the linear dsDNA with 32P and quantified the reaction products. Rad51 promoted the formation of joint molecules gradually in 3 h (Fig. 5). In the presence of HDHB, the formation of nicked circular DNA was significantly stimulated. This was accompanied by the releasing of linear ssDNA (Fig. 5A). The amount of joint molecules and nicked-circular DNA formed in the reactions was quantified. Although more nicked-circular DNA was formed in the presence of HDHB than in its absence, the amount of joint molecules decreased in parallel (Fig. 5B). The total amount of reaction products (joint molecules and nicked circular DNA) was not increased in HDHB-containing reactions as compared to control reaction, suggesting that HDHB stimulates nicked circular DNA formation by promoting the heteroduplex extension, but not the formation of joint molecules.

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