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Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths.
DNA Repair (Amst). 2005 Jun 08; 4(6):687-98.DR

Abstract

DNA double-strand breaks (DSBs) in yeast are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad51 forms nucleoprotein filaments at processed broken ends that effect strand exchange, forming heteroduplex DNA (hDNA) that gives rise to a gene conversion tract. We hypothesized that excess Rad51 would increase gene conversion tract lengths. We found that excess Rad51 reduced DSB-induced HR but did not alter tract lengths or other outcomes including rates of crossovers, break-induced replication, or chromosome loss. Thus, excess Rad51 appears to influence DSB-induced HR at an early stage. MAT heterozygosity largely mitigated the inhibitory effect of excess Rad51 on allelic HR, but not direct repeat HR. Excess Rad52 had no effect on DSB-induced HR efficiency or outcome, nor did it mitigate the dominant negative effects of excess Rad51. Excess Rad51 had little effect on DSB-induced lethality in wild-type cells, but it did enhance lethality in yku70Delta mutants. Interestingly, dnl4Delta showed marked DSB-induced lethality but this was not further enhanced by excess Rad51. The differential effects of yku70Delta and dnl4Delta indicate that the enhanced killing with excess Rad51 in yku70Delta is not due to its NHEJ defect, but may reflect its defect in end-protection and/or its inability to escape from checkpoint arrest. Srs2 displaces Rad51 from nucleoprotein filaments in vitro, suggesting that excess Rad51 might antagonize Srs2. We show that excess Rad51 does not reduce survival of wild-type cells treated with methylmethane sulfonate (MMS), or cells suffering a single DSB. In contrast, excess Rad51 sensitized srs2Delta cells to both MMS and a single DSB. These results support the idea that excess Rad51 antagonizes Srs2, and underscores the importance of displacing Rad51 from nucleoprotein filaments to achieve optimum repair efficiency.

Authors+Show Affiliations

Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15878310

Citation

Paffett, Kimberly S., et al. "Overexpression of Rad51 Inhibits Double-strand Break-induced Homologous Recombination but Does Not Affect Gene Conversion Tract Lengths." DNA Repair, vol. 4, no. 6, 2005, pp. 687-98.
Paffett KS, Clikeman JA, Palmer S, et al. Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths. DNA Repair (Amst). 2005;4(6):687-98.
Paffett, K. S., Clikeman, J. A., Palmer, S., & Nickoloff, J. A. (2005). Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths. DNA Repair, 4(6), 687-98.
Paffett KS, et al. Overexpression of Rad51 Inhibits Double-strand Break-induced Homologous Recombination but Does Not Affect Gene Conversion Tract Lengths. DNA Repair (Amst). 2005 Jun 8;4(6):687-98. PubMed PMID: 15878310.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths. AU - Paffett,Kimberly S, AU - Clikeman,Jennifer A, AU - Palmer,Sean, AU - Nickoloff,Jac A, PY - 2004/12/01/received PY - 2005/03/02/revised PY - 2005/03/03/accepted PY - 2005/5/10/pubmed PY - 2005/9/27/medline PY - 2005/5/10/entrez SP - 687 EP - 98 JF - DNA repair JO - DNA Repair (Amst) VL - 4 IS - 6 N2 - DNA double-strand breaks (DSBs) in yeast are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad51 forms nucleoprotein filaments at processed broken ends that effect strand exchange, forming heteroduplex DNA (hDNA) that gives rise to a gene conversion tract. We hypothesized that excess Rad51 would increase gene conversion tract lengths. We found that excess Rad51 reduced DSB-induced HR but did not alter tract lengths or other outcomes including rates of crossovers, break-induced replication, or chromosome loss. Thus, excess Rad51 appears to influence DSB-induced HR at an early stage. MAT heterozygosity largely mitigated the inhibitory effect of excess Rad51 on allelic HR, but not direct repeat HR. Excess Rad52 had no effect on DSB-induced HR efficiency or outcome, nor did it mitigate the dominant negative effects of excess Rad51. Excess Rad51 had little effect on DSB-induced lethality in wild-type cells, but it did enhance lethality in yku70Delta mutants. Interestingly, dnl4Delta showed marked DSB-induced lethality but this was not further enhanced by excess Rad51. The differential effects of yku70Delta and dnl4Delta indicate that the enhanced killing with excess Rad51 in yku70Delta is not due to its NHEJ defect, but may reflect its defect in end-protection and/or its inability to escape from checkpoint arrest. Srs2 displaces Rad51 from nucleoprotein filaments in vitro, suggesting that excess Rad51 might antagonize Srs2. We show that excess Rad51 does not reduce survival of wild-type cells treated with methylmethane sulfonate (MMS), or cells suffering a single DSB. In contrast, excess Rad51 sensitized srs2Delta cells to both MMS and a single DSB. These results support the idea that excess Rad51 antagonizes Srs2, and underscores the importance of displacing Rad51 from nucleoprotein filaments to achieve optimum repair efficiency. SN - 1568-7864 UR - https://www.unboundmedicine.com/medline/citation/15878310/Overexpression_of_Rad51_inhibits_double_strand_break_induced_homologous_recombination_but_does_not_affect_gene_conversion_tract_lengths_ DB - PRIME DP - Unbound Medicine ER -