A Double-Strand Break within a Yeast Artificial Chromosome (YAC) Containing Human DNA Can Result in YAC Loss, Deletion, or Cell Lethality

Abstract

Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G 2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.

Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group

“…By introducing NHEJ, cells of higher eukaryotes rapidly restore integrity in their large genome, relying on HRR only to restore critical DNA sequences at a later time. This controlled approach to HRR may reduce illegitimate recombination events that could be initiated by repeat sequences (Bennett et al, 1996) and may explain the suppression observed in the utilization of HRR for the initial rejoining event (see below).…”

“…It is possible that down-regulation of HRR in vertebrate cells aims at reducing the occurrence of illegitimate recombination events supported by repeated sequences in the presence of DSBs (Bennett et al, 1996(Bennett et al, , 1997. To counteract associated defects on DNA DSB repair, DNA-PKcs, an enzyme not present in yeast, may have evolved in vertebrate cells to accelerate NHEJ.…”

Double-strand DNA Break Formation Mediated by Flap Endonuclease-1

“…Thus, even in the presence of various mechanisms preventing DSB formation from closely spaced DNA damages on opposing DNA strands, there is still a risk of DSB-induced cytotoxicity, as only a small number of DSBs is sufficient to kill a cell (36). Such DNA damages can be found within DNA damage clusters induced by ionizing radiations (2).…”

Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

“…As illustrated in Figure 1, we assume that the burst size decreases when a double-strand break of one of the coinfecting phages remains unrepaired. This assumption is based on the experimental evidence that a single unrepaired double-strand break on a plasmid molecule or a yeast artificial chromosome induces lethality to a cell (Bennett et al 1993(Bennett et al , 1996. We thus introduce another parameter of burst size under induction of damage to the host cell physiology b 1 , which is less than or equal to default burst size b 0 .…”