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RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.

Westmoreland J, Ma W, Yan Y, Van Hulle K, Malkova A, Resnick MA - PLoS Genet. (2009)

Bottom Line: However, in rad50 and mre11 mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M.Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair.A similar requirement was found for RAD50 in asynchronously growing cells.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Stability Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

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Changes in broken chromosomes following irradiation of HR defective mutants.(A) Absence of repair in HR defective mutants. Logarithmically growing rad51Δ, rad50Δ, and rad52Δ cells were arrested in G2/M with nocodazole, irradiated with 80 krad, and returned to growth medium for up to 4 hrs (as described in Figure 1A). A single break in Chr III resulted in a band above the bottom doublet (230 kb) at 0 hrs. While there was no evidence of repair, the rad51Δ strain, unlike rad50Δ and rad52Δ, exhibited limited repair of ChrXII, half of which contains ribosomal DNA (first band below the wells). Chromosomal material was displayed by PFGE (CHEF, see Materials and Methods) and the gels were stained with SybrGold. (B) PFGE-shift of broken fragments in rad52Δ but not in rad50Δ. Cells of rad50Δ and rad51Δ were arrested in G2/M, irradiated, and returned to growth medium as described in Figure 2A. Samples were run on a pulsed-field gel (TAFE; see Materials and Methods), and a southern transfer was hybridized to a probe (V16) specific for unique sequence near the left telomere of Chr V (16 kb from the end). The lower portion of the smear of broken fragments below the intact full-length Chr V shifts upward by 0.5 hours in the rad52Δ but not the rad50Δ strain.
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pgen-1000656-g002: Changes in broken chromosomes following irradiation of HR defective mutants.(A) Absence of repair in HR defective mutants. Logarithmically growing rad51Δ, rad50Δ, and rad52Δ cells were arrested in G2/M with nocodazole, irradiated with 80 krad, and returned to growth medium for up to 4 hrs (as described in Figure 1A). A single break in Chr III resulted in a band above the bottom doublet (230 kb) at 0 hrs. While there was no evidence of repair, the rad51Δ strain, unlike rad50Δ and rad52Δ, exhibited limited repair of ChrXII, half of which contains ribosomal DNA (first band below the wells). Chromosomal material was displayed by PFGE (CHEF, see Materials and Methods) and the gels were stained with SybrGold. (B) PFGE-shift of broken fragments in rad52Δ but not in rad50Δ. Cells of rad50Δ and rad51Δ were arrested in G2/M, irradiated, and returned to growth medium as described in Figure 2A. Samples were run on a pulsed-field gel (TAFE; see Materials and Methods), and a southern transfer was hybridized to a probe (V16) specific for unique sequence near the left telomere of Chr V (16 kb from the end). The lower portion of the smear of broken fragments below the intact full-length Chr V shifts upward by 0.5 hours in the rad52Δ but not the rad50Δ strain.

Mentions: There is no detectable restitution of chromosomes in the rad50Δ, rad52Δ, and rad51Δ mutants up to 4 hours after 80 krad exposure of G2/M cells (Figure 2A and analysis of PFGE gels; data not shown). Recombinational mechanisms account for nearly all repair of radiation induced DSBs since a mutation in the DNL4 gene, required for end-joining ligation, failed to influence the postirradiation status of DSBs or survival in a rad52 or WT background (data not shown) consistent with results obtained with ku70 rad52 vs rad52 logarithmically growing cells [28]. There appears to be some repair of chromosome XII in the rad51Δ mutant, which is presumably due to single-strand annealing repair of resected, ends (see below) involving the ribosomal repeats on this chromosome. The overall lack of restitution of full size chromosomes in the rad− mutants was confirmed by Southern analysis of individual chromosomes following irradiation and incubation (presented in Figure 2B is Chr V). The small amount of restitution in the rad52 and rad51 mutants (data not shown) shortly after irradiation is being investigated. While not due to endjoining (data not shown for dnl4 rad52), it might be due to repair of closely-opposed single-strand breaks [26].


RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.

Westmoreland J, Ma W, Yan Y, Van Hulle K, Malkova A, Resnick MA - PLoS Genet. (2009)

Changes in broken chromosomes following irradiation of HR defective mutants.(A) Absence of repair in HR defective mutants. Logarithmically growing rad51Δ, rad50Δ, and rad52Δ cells were arrested in G2/M with nocodazole, irradiated with 80 krad, and returned to growth medium for up to 4 hrs (as described in Figure 1A). A single break in Chr III resulted in a band above the bottom doublet (230 kb) at 0 hrs. While there was no evidence of repair, the rad51Δ strain, unlike rad50Δ and rad52Δ, exhibited limited repair of ChrXII, half of which contains ribosomal DNA (first band below the wells). Chromosomal material was displayed by PFGE (CHEF, see Materials and Methods) and the gels were stained with SybrGold. (B) PFGE-shift of broken fragments in rad52Δ but not in rad50Δ. Cells of rad50Δ and rad51Δ were arrested in G2/M, irradiated, and returned to growth medium as described in Figure 2A. Samples were run on a pulsed-field gel (TAFE; see Materials and Methods), and a southern transfer was hybridized to a probe (V16) specific for unique sequence near the left telomere of Chr V (16 kb from the end). The lower portion of the smear of broken fragments below the intact full-length Chr V shifts upward by 0.5 hours in the rad52Δ but not the rad50Δ strain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2734177&req=5

pgen-1000656-g002: Changes in broken chromosomes following irradiation of HR defective mutants.(A) Absence of repair in HR defective mutants. Logarithmically growing rad51Δ, rad50Δ, and rad52Δ cells were arrested in G2/M with nocodazole, irradiated with 80 krad, and returned to growth medium for up to 4 hrs (as described in Figure 1A). A single break in Chr III resulted in a band above the bottom doublet (230 kb) at 0 hrs. While there was no evidence of repair, the rad51Δ strain, unlike rad50Δ and rad52Δ, exhibited limited repair of ChrXII, half of which contains ribosomal DNA (first band below the wells). Chromosomal material was displayed by PFGE (CHEF, see Materials and Methods) and the gels were stained with SybrGold. (B) PFGE-shift of broken fragments in rad52Δ but not in rad50Δ. Cells of rad50Δ and rad51Δ were arrested in G2/M, irradiated, and returned to growth medium as described in Figure 2A. Samples were run on a pulsed-field gel (TAFE; see Materials and Methods), and a southern transfer was hybridized to a probe (V16) specific for unique sequence near the left telomere of Chr V (16 kb from the end). The lower portion of the smear of broken fragments below the intact full-length Chr V shifts upward by 0.5 hours in the rad52Δ but not the rad50Δ strain.
Mentions: There is no detectable restitution of chromosomes in the rad50Δ, rad52Δ, and rad51Δ mutants up to 4 hours after 80 krad exposure of G2/M cells (Figure 2A and analysis of PFGE gels; data not shown). Recombinational mechanisms account for nearly all repair of radiation induced DSBs since a mutation in the DNL4 gene, required for end-joining ligation, failed to influence the postirradiation status of DSBs or survival in a rad52 or WT background (data not shown) consistent with results obtained with ku70 rad52 vs rad52 logarithmically growing cells [28]. There appears to be some repair of chromosome XII in the rad51Δ mutant, which is presumably due to single-strand annealing repair of resected, ends (see below) involving the ribosomal repeats on this chromosome. The overall lack of restitution of full size chromosomes in the rad− mutants was confirmed by Southern analysis of individual chromosomes following irradiation and incubation (presented in Figure 2B is Chr V). The small amount of restitution in the rad52 and rad51 mutants (data not shown) shortly after irradiation is being investigated. While not due to endjoining (data not shown for dnl4 rad52), it might be due to repair of closely-opposed single-strand breaks [26].

Bottom Line: However, in rad50 and mre11 mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M.Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair.A similar requirement was found for RAD50 in asynchronously growing cells.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Stability Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

Show MeSH
Related in: MedlinePlus