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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 stimulates 5′-3′ Rad51-mediated heteroduplex extension.(A) Diagram of strand exchange reaction. Jm, joint molecule; nc, nicked-circular DNA. (B) hRad51 purification. (C) DNA-end requirements for hRad51-catalyzed strand exchange. Different linear dsDNA substrates as indicated were used in the strand exchange reactions supplied with 100 mM (NH4)2SO4. The gray strand of each dsDNA substrate is the strand complementary to the circular ssDNA. The reactions were stopped 2 h after the reactions were initiated. (D), (E) Strand exchange reactions were performed in the presence of 60 mM KCl and 2 mM CaCl2. dsDNA with 3′-overhanging termini (D) or 5′-overhanging termini (E) was used in the reaction. An annealed nicked-circular DNA marker is in lane 12. HDHB concentration in the reactions was: lanes 3 and 8, 50 nM; lanes 4 and 9, 100 nM; lanes 5 and 10, 150 nM. Quantification of nicked-circular DNA formed in the reaction was shown on the bottom of each gel. (F) Strand exchange reactions were performed in the presence of 50 mM (NH4)2SO4. dsDNA with 3′-overhanging termini was used. (G) Reaction was performed with dsDNA with blunt-end termini or recessed end. HDHB concentration was 100 nM. (H) Walker B mutant HDHB did not promote heteroduplex extension. The concentration of wild-type or mutant HDHB was 100 nM.
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pone.0116852.g004: HDHB stimulates 5′-3′ Rad51-mediated heteroduplex extension.(A) Diagram of strand exchange reaction. Jm, joint molecule; nc, nicked-circular DNA. (B) hRad51 purification. (C) DNA-end requirements for hRad51-catalyzed strand exchange. Different linear dsDNA substrates as indicated were used in the strand exchange reactions supplied with 100 mM (NH4)2SO4. The gray strand of each dsDNA substrate is the strand complementary to the circular ssDNA. The reactions were stopped 2 h after the reactions were initiated. (D), (E) Strand exchange reactions were performed in the presence of 60 mM KCl and 2 mM CaCl2. dsDNA with 3′-overhanging termini (D) or 5′-overhanging termini (E) was used in the reaction. An annealed nicked-circular DNA marker is in lane 12. HDHB concentration in the reactions was: lanes 3 and 8, 50 nM; lanes 4 and 9, 100 nM; lanes 5 and 10, 150 nM. Quantification of nicked-circular DNA formed in the reaction was shown on the bottom of each gel. (F) Strand exchange reactions were performed in the presence of 50 mM (NH4)2SO4. dsDNA with 3′-overhanging termini was used. (G) Reaction was performed with dsDNA with blunt-end termini or recessed end. HDHB concentration was 100 nM. (H) Walker B mutant HDHB did not promote heteroduplex extension. The concentration of wild-type or mutant HDHB was 100 nM.

Mentions: We investigated Rad51-mediated strand exchange in the presence of HDHB. Human Rad51 (hRad51) was purified as described (Fig. 4B) [34]. An in vitro strand-exchange reaction between linear X174 dsDNA and circular ssDNA catalyzed by hRad51 was performed (Fig. 4A) in the presence of either (NH4)2SO4 or CaCl2 [35], [36], [37], [38]. We firstly tested the end requirements for DNA substrates in the strand-exchange reaction. Consistent with previous findings in yeast [39], [40], [41], hRad51 only promoted strand exchange in the reactions containing dsDNAs with at least one overhanging ssDNA tail on the complementary strand (Fig. 4C).


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 stimulates 5′-3′ Rad51-mediated heteroduplex extension.(A) Diagram of strand exchange reaction. Jm, joint molecule; nc, nicked-circular DNA. (B) hRad51 purification. (C) DNA-end requirements for hRad51-catalyzed strand exchange. Different linear dsDNA substrates as indicated were used in the strand exchange reactions supplied with 100 mM (NH4)2SO4. The gray strand of each dsDNA substrate is the strand complementary to the circular ssDNA. The reactions were stopped 2 h after the reactions were initiated. (D), (E) Strand exchange reactions were performed in the presence of 60 mM KCl and 2 mM CaCl2. dsDNA with 3′-overhanging termini (D) or 5′-overhanging termini (E) was used in the reaction. An annealed nicked-circular DNA marker is in lane 12. HDHB concentration in the reactions was: lanes 3 and 8, 50 nM; lanes 4 and 9, 100 nM; lanes 5 and 10, 150 nM. Quantification of nicked-circular DNA formed in the reaction was shown on the bottom of each gel. (F) Strand exchange reactions were performed in the presence of 50 mM (NH4)2SO4. dsDNA with 3′-overhanging termini was used. (G) Reaction was performed with dsDNA with blunt-end termini or recessed end. HDHB concentration was 100 nM. (H) Walker B mutant HDHB did not promote heteroduplex extension. The concentration of wild-type or mutant HDHB was 100 nM.
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pone.0116852.g004: HDHB stimulates 5′-3′ Rad51-mediated heteroduplex extension.(A) Diagram of strand exchange reaction. Jm, joint molecule; nc, nicked-circular DNA. (B) hRad51 purification. (C) DNA-end requirements for hRad51-catalyzed strand exchange. Different linear dsDNA substrates as indicated were used in the strand exchange reactions supplied with 100 mM (NH4)2SO4. The gray strand of each dsDNA substrate is the strand complementary to the circular ssDNA. The reactions were stopped 2 h after the reactions were initiated. (D), (E) Strand exchange reactions were performed in the presence of 60 mM KCl and 2 mM CaCl2. dsDNA with 3′-overhanging termini (D) or 5′-overhanging termini (E) was used in the reaction. An annealed nicked-circular DNA marker is in lane 12. HDHB concentration in the reactions was: lanes 3 and 8, 50 nM; lanes 4 and 9, 100 nM; lanes 5 and 10, 150 nM. Quantification of nicked-circular DNA formed in the reaction was shown on the bottom of each gel. (F) Strand exchange reactions were performed in the presence of 50 mM (NH4)2SO4. dsDNA with 3′-overhanging termini was used. (G) Reaction was performed with dsDNA with blunt-end termini or recessed end. HDHB concentration was 100 nM. (H) Walker B mutant HDHB did not promote heteroduplex extension. The concentration of wild-type or mutant HDHB was 100 nM.
Mentions: We investigated Rad51-mediated strand exchange in the presence of HDHB. Human Rad51 (hRad51) was purified as described (Fig. 4B) [34]. An in vitro strand-exchange reaction between linear X174 dsDNA and circular ssDNA catalyzed by hRad51 was performed (Fig. 4A) in the presence of either (NH4)2SO4 or CaCl2 [35], [36], [37], [38]. We firstly tested the end requirements for DNA substrates in the strand-exchange reaction. Consistent with previous findings in yeast [39], [40], [41], hRad51 only promoted strand exchange in the reactions containing dsDNAs with at least one overhanging ssDNA tail on the complementary strand (Fig. 4C).

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