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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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The PFGE-shift associated with a random γ-induced single DSB after low doses or HO-endonuclease produced DSBs.(A) PFGE-shift associated with a single radiation-induced DSB in circular Chr V in WT but not rad50Δ cells. Nocodazole arrested G2/M cells containing a circular chromosome V were irradiated with 40 krads and returned to YPDA media. Plugs were prepared at the indicated times and run on PFGE (TAFE). Southern transfers were hybridized with a MET6 probe specific for the 530 kb Chr V. Results are similar to those obtained using circular Chr III, except the resected form of the putative linear dimer band (D**) seen at ∼1080 krads in WT (left image, 1 hr) is not well-resolved from the unresected form, twice the molecular weight of the non-resected monomer band (M). PFGE-shift of the linearized monomer band in WT reaches a maximum at an apparent molecular weight of ∼“600” kb at 1 hr, after which the M** band mostly disappears due to repair and recircularization. In the rad50Δ strain the non-resected monomer band (M) persists for 4 hrs with only faint smearing above it as also seen in rad50Δ using the circular Chr III construct (Figure 5A). The putative linear dimer band (D) was also detected with rad50Δ cells. (B) Resection events at low dose suggest one- and two-ended events. Arrested rad50Δ and rad52Δ G2/M cells containing circular Chr III were irradiated with 20 krads and returned to YPDA media. Plugs were prepared at the indicated time points and run on PFGE using CHEF analysis (see Materials and Methods). Two PFGE-shift bands, M* (at 0.5 to 4 hrs) and M** (at 4 hrs) were detected. As suggested in the text, the M* band is consistent with linearized molecules that were resected at only one end of the DSB. The M** seen in rad50Δ at 4 hrs is at the same maximum shift position as the M** band typically seen in rad52Δ by 2 hrs (see right group of lanes) and is proposed to be due to molecules that were resected at both ends with 3′ single-strand tails long enough to cause maximum shift. The persistence of significant portions of non-resected and of partially resected molecules in rad50Δ but not in rad52Δ demonstrates that RAD50 is required for the rapid and efficient initiation of resection at damaged ends in WT and rad52Δ cells. (C) Lack of resection at HO-induced DSB in rad50Δ cells is reflected by absence of PFGE-shift. Resection was analyzed in the nocodazole-arrested rad50Δ cells (MN108) using procedures similar to those described in Figure 3B. Unlike for WT (Figure 3B), there was very little PFGE-shift, even at 5 hr.
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pgen-1000656-g006: The PFGE-shift associated with a random γ-induced single DSB after low doses or HO-endonuclease produced DSBs.(A) PFGE-shift associated with a single radiation-induced DSB in circular Chr V in WT but not rad50Δ cells. Nocodazole arrested G2/M cells containing a circular chromosome V were irradiated with 40 krads and returned to YPDA media. Plugs were prepared at the indicated times and run on PFGE (TAFE). Southern transfers were hybridized with a MET6 probe specific for the 530 kb Chr V. Results are similar to those obtained using circular Chr III, except the resected form of the putative linear dimer band (D**) seen at ∼1080 krads in WT (left image, 1 hr) is not well-resolved from the unresected form, twice the molecular weight of the non-resected monomer band (M). PFGE-shift of the linearized monomer band in WT reaches a maximum at an apparent molecular weight of ∼“600” kb at 1 hr, after which the M** band mostly disappears due to repair and recircularization. In the rad50Δ strain the non-resected monomer band (M) persists for 4 hrs with only faint smearing above it as also seen in rad50Δ using the circular Chr III construct (Figure 5A). The putative linear dimer band (D) was also detected with rad50Δ cells. (B) Resection events at low dose suggest one- and two-ended events. Arrested rad50Δ and rad52Δ G2/M cells containing circular Chr III were irradiated with 20 krads and returned to YPDA media. Plugs were prepared at the indicated time points and run on PFGE using CHEF analysis (see Materials and Methods). Two PFGE-shift bands, M* (at 0.5 to 4 hrs) and M** (at 4 hrs) were detected. As suggested in the text, the M* band is consistent with linearized molecules that were resected at only one end of the DSB. The M** seen in rad50Δ at 4 hrs is at the same maximum shift position as the M** band typically seen in rad52Δ by 2 hrs (see right group of lanes) and is proposed to be due to molecules that were resected at both ends with 3′ single-strand tails long enough to cause maximum shift. The persistence of significant portions of non-resected and of partially resected molecules in rad50Δ but not in rad52Δ demonstrates that RAD50 is required for the rapid and efficient initiation of resection at damaged ends in WT and rad52Δ cells. (C) Lack of resection at HO-induced DSB in rad50Δ cells is reflected by absence of PFGE-shift. Resection was analyzed in the nocodazole-arrested rad50Δ cells (MN108) using procedures similar to those described in Figure 3B. Unlike for WT (Figure 3B), there was very little PFGE-shift, even at 5 hr.

Mentions: Events in the rad50Δ mutant after γ-linearization of circular Chr III differ dramatically from WT and the rad51Δ and rad52Δ mutants. Rather than a rapid accumulation of the shifted ∼430 kb band, there is a slight “smear” in only ∼20% of the molecules beginning at about 1 hr after an 80 krad dose (compare Figure 5A with Figures 4A and 4C). Similar results were found for another circular chromosome, Chr V (540 kb; Figure 6A) following a dose of 40 krad. (Because of the nearly two-fold increase in size, the dose was reduced two-fold to induce a comparable number of broken chromosomes.) As shown in Figure 5B, differences in genetic control of the appearance of shifted molecules are even more distinct using CHEF gel conditions (see Materials and Methods for TAFE vs CHEF procedures). Treatment of the plugs from the rad50Δ mutant with MBN decreases the slight smear of the linear Chr III band, indicating that the shifted material is due to resection (Figure 5C). Based on the persistence of a large portion of unshifted and, therefore, unresected 300 kb molecules from the rad50Δ cells, the MRX complex is concluded to be essential for the rapid initiation of resection of radiation induced DSBs in WT, rad52 and -51 G2/M arrested cells (also growing cells; data not shown).


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)

The PFGE-shift associated with a random γ-induced single DSB after low doses or HO-endonuclease produced DSBs.(A) PFGE-shift associated with a single radiation-induced DSB in circular Chr V in WT but not rad50Δ cells. Nocodazole arrested G2/M cells containing a circular chromosome V were irradiated with 40 krads and returned to YPDA media. Plugs were prepared at the indicated times and run on PFGE (TAFE). Southern transfers were hybridized with a MET6 probe specific for the 530 kb Chr V. Results are similar to those obtained using circular Chr III, except the resected form of the putative linear dimer band (D**) seen at ∼1080 krads in WT (left image, 1 hr) is not well-resolved from the unresected form, twice the molecular weight of the non-resected monomer band (M). PFGE-shift of the linearized monomer band in WT reaches a maximum at an apparent molecular weight of ∼“600” kb at 1 hr, after which the M** band mostly disappears due to repair and recircularization. In the rad50Δ strain the non-resected monomer band (M) persists for 4 hrs with only faint smearing above it as also seen in rad50Δ using the circular Chr III construct (Figure 5A). The putative linear dimer band (D) was also detected with rad50Δ cells. (B) Resection events at low dose suggest one- and two-ended events. Arrested rad50Δ and rad52Δ G2/M cells containing circular Chr III were irradiated with 20 krads and returned to YPDA media. Plugs were prepared at the indicated time points and run on PFGE using CHEF analysis (see Materials and Methods). Two PFGE-shift bands, M* (at 0.5 to 4 hrs) and M** (at 4 hrs) were detected. As suggested in the text, the M* band is consistent with linearized molecules that were resected at only one end of the DSB. The M** seen in rad50Δ at 4 hrs is at the same maximum shift position as the M** band typically seen in rad52Δ by 2 hrs (see right group of lanes) and is proposed to be due to molecules that were resected at both ends with 3′ single-strand tails long enough to cause maximum shift. The persistence of significant portions of non-resected and of partially resected molecules in rad50Δ but not in rad52Δ demonstrates that RAD50 is required for the rapid and efficient initiation of resection at damaged ends in WT and rad52Δ cells. (C) Lack of resection at HO-induced DSB in rad50Δ cells is reflected by absence of PFGE-shift. Resection was analyzed in the nocodazole-arrested rad50Δ cells (MN108) using procedures similar to those described in Figure 3B. Unlike for WT (Figure 3B), there was very little PFGE-shift, even at 5 hr.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734177&req=5

pgen-1000656-g006: The PFGE-shift associated with a random γ-induced single DSB after low doses or HO-endonuclease produced DSBs.(A) PFGE-shift associated with a single radiation-induced DSB in circular Chr V in WT but not rad50Δ cells. Nocodazole arrested G2/M cells containing a circular chromosome V were irradiated with 40 krads and returned to YPDA media. Plugs were prepared at the indicated times and run on PFGE (TAFE). Southern transfers were hybridized with a MET6 probe specific for the 530 kb Chr V. Results are similar to those obtained using circular Chr III, except the resected form of the putative linear dimer band (D**) seen at ∼1080 krads in WT (left image, 1 hr) is not well-resolved from the unresected form, twice the molecular weight of the non-resected monomer band (M). PFGE-shift of the linearized monomer band in WT reaches a maximum at an apparent molecular weight of ∼“600” kb at 1 hr, after which the M** band mostly disappears due to repair and recircularization. In the rad50Δ strain the non-resected monomer band (M) persists for 4 hrs with only faint smearing above it as also seen in rad50Δ using the circular Chr III construct (Figure 5A). The putative linear dimer band (D) was also detected with rad50Δ cells. (B) Resection events at low dose suggest one- and two-ended events. Arrested rad50Δ and rad52Δ G2/M cells containing circular Chr III were irradiated with 20 krads and returned to YPDA media. Plugs were prepared at the indicated time points and run on PFGE using CHEF analysis (see Materials and Methods). Two PFGE-shift bands, M* (at 0.5 to 4 hrs) and M** (at 4 hrs) were detected. As suggested in the text, the M* band is consistent with linearized molecules that were resected at only one end of the DSB. The M** seen in rad50Δ at 4 hrs is at the same maximum shift position as the M** band typically seen in rad52Δ by 2 hrs (see right group of lanes) and is proposed to be due to molecules that were resected at both ends with 3′ single-strand tails long enough to cause maximum shift. The persistence of significant portions of non-resected and of partially resected molecules in rad50Δ but not in rad52Δ demonstrates that RAD50 is required for the rapid and efficient initiation of resection at damaged ends in WT and rad52Δ cells. (C) Lack of resection at HO-induced DSB in rad50Δ cells is reflected by absence of PFGE-shift. Resection was analyzed in the nocodazole-arrested rad50Δ cells (MN108) using procedures similar to those described in Figure 3B. Unlike for WT (Figure 3B), there was very little PFGE-shift, even at 5 hr.
Mentions: Events in the rad50Δ mutant after γ-linearization of circular Chr III differ dramatically from WT and the rad51Δ and rad52Δ mutants. Rather than a rapid accumulation of the shifted ∼430 kb band, there is a slight “smear” in only ∼20% of the molecules beginning at about 1 hr after an 80 krad dose (compare Figure 5A with Figures 4A and 4C). Similar results were found for another circular chromosome, Chr V (540 kb; Figure 6A) following a dose of 40 krad. (Because of the nearly two-fold increase in size, the dose was reduced two-fold to induce a comparable number of broken chromosomes.) As shown in Figure 5B, differences in genetic control of the appearance of shifted molecules are even more distinct using CHEF gel conditions (see Materials and Methods for TAFE vs CHEF procedures). Treatment of the plugs from the rad50Δ mutant with MBN decreases the slight smear of the linear Chr III band, indicating that the shifted material is due to resection (Figure 5C). Based on the persistence of a large portion of unshifted and, therefore, unresected 300 kb molecules from the rad50Δ cells, the MRX complex is concluded to be essential for the rapid initiation of resection of radiation induced DSBs in WT, rad52 and -51 G2/M arrested cells (also growing cells; data not shown).

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