Blunt-ended DNA double-strand breaks induced by endonucleases PvuII and EcoRV are poor substrates for repair in Saccharomyces cerevisiae.
Most mechanistic studies of repair of DNA double-strand breaks (DSBs) produced by in vivo expression of endonucleases have utilized enzymes that produce cohesive-ended DSBs such as HO, I-SceI and EcoRI. We have developed systems for expression of PvuII and EcoRV, nucleases that produce DSBs containing blunt ends, using a modified GAL1 promoter that has reduced basal activity. Expression of PvuII and EcoRV caused growth inhibition and strong cell killing in both haploid and diploid yeast cells. Surprisingly, there was little difference in sensitivities of wildtype cells and mutants defective in homologous recombination, nonhomologous end-joining (NHEJ), or both pathways. Physical analysis using standard and pulsed field gel electrophoresis demonstrated time-dependent breakage of chromosomal DNA within cells. Although ionizing radiation-induced DSBs were largely repaired within 4h, no repair of PvuII-induced breaks could be detected in diploid cells, even after arrest in G2/M. Rare survivors of PvuII expression had an increased frequency of chromosome XII deletions, an indication that a fraction of the induced DSBs could be repaired by an error-prone process. These results indicate that, unlike DSBs with complementary single-stranded DNA overhangs, blunt-ended DSBs in yeast chromosomes are poor substrates for repair by either NHEJ or recombination.
Westmoreland
James W
JW
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Summers
Jennifer A
JA
Holland
Cory L
CL
Resnick
Michael A
MA
Lewis
L Kevin
LK
D002470:Cell Survival; D053903:DNA Breaks, Double-Stranded; D053938:DNA Fragmentation; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004171:Diploidy; D015870:Gene Expression; D006238:Haploidy; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D004271:DNA, Fungal; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases