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Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis.

Che J, Smith S, Kim YJ, Shim EY, Myung K, Lee SE - PLoS Genet. (2015)

Bottom Line: Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR).Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants.Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America; Program of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

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Rad52-GFP foci in hst3Δ hst4Δ mrc1Δ and pol32Δ mrc1Δ cells persist after HU treatment.A, Flowchart showing experimental procedure. B, Representative images for different cells showing persistent Rad52-GFP foci at 8 hours after recovery from HU treatment. Size bar, 15 µm. C, Quantification of percentage of Rad52-GFP foci in the cell population. At least 300 individual cells were counted for each genotype and time point.
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pgen.1004990.g004: Rad52-GFP foci in hst3Δ hst4Δ mrc1Δ and pol32Δ mrc1Δ cells persist after HU treatment.A, Flowchart showing experimental procedure. B, Representative images for different cells showing persistent Rad52-GFP foci at 8 hours after recovery from HU treatment. Size bar, 15 µm. C, Quantification of percentage of Rad52-GFP foci in the cell population. At least 300 individual cells were counted for each genotype and time point.

Mentions: The lethality upon acute HU treatment in hst3Δ hst4Δ mrc1∆ cells could be due to an inability to repair a collapsed fork or alternatively to a more extensive fork collapse. It has been shown that HU treatment induces Rad52-green fluorescent protein (GFP) focus formation in mrc1Δ but not in wild-type nuclei, likely marking the site of stressed and collapsed replication forks and their repair [51]. We thus monitored the kinetics of Rad52-GFP foci as a surrogate for the repair of collapsed replication forks in order to dissect HU-induced fork collapse in mrc1∆ cells. We found that Rad52-GFP foci appeared in mrc1Δ cells upon HU treatment, reached a maximum level after releasing cells into fresh YEPD medium, and gradually disappeared most likely due to repair (Fig. 4). Importantly, in hst3Δ hst4Δ mrc1Δ cells and pol32Δ mrc1Δ cells, the Rad52-GFP foci persisted for up to 8 h after HU treatment, suggesting that hst3Δ hst4Δ or pol32Δ cells are deficient in repairing collapsed replication forks (Fig. 4). To test if the repair deficiency in hst3Δ hst4Δ mrc1Δ mutants could be attributed to hyper-acetylation of H3K56, we deleted ASF1 and monitored Rad52-GFP kinetics following HU treatment. Deletion of ASF1 led to a high level of Rad52-GFP foci at 6–8 h after release from HU, consistent with the role of Asf1 in fork stability [52], yet significantly offset the level of Rad52-GFP in hst3Δ hst4Δ mrc1Δ cells (Fig. 4A and B). Deletion of HST3 and HST4 also led to an increase in spontaneous Rad52-GFP focus formation and sensitivity to acute HU treatment in the BY4741 strain, but not in another genetic background (JKM139, see Fig. 4A and C, and S6 Fig.). We surmise that Hst3 and Hst4 contribute to the integrity of replication forks in certain genetic backgrounds. The results support the role of Hst3, Hst4 and Pol32 in the repair of collapsed replication forks.


Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis.

Che J, Smith S, Kim YJ, Shim EY, Myung K, Lee SE - PLoS Genet. (2015)

Rad52-GFP foci in hst3Δ hst4Δ mrc1Δ and pol32Δ mrc1Δ cells persist after HU treatment.A, Flowchart showing experimental procedure. B, Representative images for different cells showing persistent Rad52-GFP foci at 8 hours after recovery from HU treatment. Size bar, 15 µm. C, Quantification of percentage of Rad52-GFP foci in the cell population. At least 300 individual cells were counted for each genotype and time point.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4338291&req=5

pgen.1004990.g004: Rad52-GFP foci in hst3Δ hst4Δ mrc1Δ and pol32Δ mrc1Δ cells persist after HU treatment.A, Flowchart showing experimental procedure. B, Representative images for different cells showing persistent Rad52-GFP foci at 8 hours after recovery from HU treatment. Size bar, 15 µm. C, Quantification of percentage of Rad52-GFP foci in the cell population. At least 300 individual cells were counted for each genotype and time point.
Mentions: The lethality upon acute HU treatment in hst3Δ hst4Δ mrc1∆ cells could be due to an inability to repair a collapsed fork or alternatively to a more extensive fork collapse. It has been shown that HU treatment induces Rad52-green fluorescent protein (GFP) focus formation in mrc1Δ but not in wild-type nuclei, likely marking the site of stressed and collapsed replication forks and their repair [51]. We thus monitored the kinetics of Rad52-GFP foci as a surrogate for the repair of collapsed replication forks in order to dissect HU-induced fork collapse in mrc1∆ cells. We found that Rad52-GFP foci appeared in mrc1Δ cells upon HU treatment, reached a maximum level after releasing cells into fresh YEPD medium, and gradually disappeared most likely due to repair (Fig. 4). Importantly, in hst3Δ hst4Δ mrc1Δ cells and pol32Δ mrc1Δ cells, the Rad52-GFP foci persisted for up to 8 h after HU treatment, suggesting that hst3Δ hst4Δ or pol32Δ cells are deficient in repairing collapsed replication forks (Fig. 4). To test if the repair deficiency in hst3Δ hst4Δ mrc1Δ mutants could be attributed to hyper-acetylation of H3K56, we deleted ASF1 and monitored Rad52-GFP kinetics following HU treatment. Deletion of ASF1 led to a high level of Rad52-GFP foci at 6–8 h after release from HU, consistent with the role of Asf1 in fork stability [52], yet significantly offset the level of Rad52-GFP in hst3Δ hst4Δ mrc1Δ cells (Fig. 4A and B). Deletion of HST3 and HST4 also led to an increase in spontaneous Rad52-GFP focus formation and sensitivity to acute HU treatment in the BY4741 strain, but not in another genetic background (JKM139, see Fig. 4A and C, and S6 Fig.). We surmise that Hst3 and Hst4 contribute to the integrity of replication forks in certain genetic backgrounds. The results support the role of Hst3, Hst4 and Pol32 in the repair of collapsed replication forks.

Bottom Line: Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR).Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants.Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America; Program of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

Show MeSH
Related in: MedlinePlus