Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk)

No abstract

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.

To initially determine the effect that basepair mismatch has on homologous recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2. These tk genes are =81 % homologous at the nucleotide level. We observed that, in mouse LTK-cells, intrachromosomal recombination between type 1 and type 2 tk sequences is reduced by a factor of at least 1000 relative to the rate of intrachromosomal recombination between homologous type 1 tk sequences. In sharp contrast, the rate of intermolecular or intramolecular extrachromosomal recombination between the heterologous tk sequences introduced by calcium phosphate or microinjection was reduced only by a factor of 3 to 15 compared with extrachromosomal homologous tk crosses. Our results suggest differences between the mechanisms of extrachromosomal and intrachromosomal recombination in mammalian cells.To elucidate the mechanism of homologous recombination in mammalian cells, investigators have studied genetic recombination using both extrachromosomal (1-10) and intrachromosomal (1, 11-14) systems. One important issue to address regarding homologous recombination is how the extent of homology between sequences influences the rate of recombination. Two groups have addressed this issue using extrachromosomal mammalian recombination systems (15, 16), whereas our laboratory has used an intrachromosomal system (17). The general finding is that when two sequences share several hundred base pairs (bp) of homology, the rate of recombination is proportional to the amount of homology. When the homology is reduced below -200 bp, the recombination rate drops off rapidly. In contrast to mammalian homologous recombination, prokaryotic recombination appears to require only -50 bp of homology to proceed efficiently (18)(19)(20).In each of the above studies of the homology requirements of mammalian recombination, the rate of recombination was determined as a function of the extent of sequence overlap. Another way to study the homology requirements of recombination is to determine the effect that base-pair mismatch has on the recombination rate. It has been shown that the homologous recombination machinery of Escherichia coli is very sensitive to base-pair mismatch, with 16% mismatch resulting in a decrease by a factor of 100 in the rate of phage-plasmid recombination (19). In E. coli, the crucial factor that determines the recombination rate appears to be the length of uninterrupted stretches of homology (19,20). In mammalian cells, it has been shown that recombination between the genomes of adenovirus types 2 and 5 can occur within regions exhibiting 90% sequence homology, but not within regions exhibiting 47% sequence homology (21).As a further step toward understanding the homology requirements of recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2 (HSV-1 and HSV-2, respectively). These tk genes are ...

In response to DNA double strand breaks, the histone variant H2AX at the break site is phosphorylated at serine 139 by DNA damage sensor kinases such as ataxia telangiectasia-mutated, forming ␥-H2AX. This phosphorylation event is critical for sustained recruitment of other proteins to repair the break. After repair, restoration of the cell to a prestress state is associated with ␥-H2AX dephosphorylation and dissolution of ␥-H2AX-associated damage foci. The phosphatases PP2A and PP4 have previously been shown to dephosphorylate ␥-H2AX. Here, we demonstrate that the wild-type p53-induced phosphatase 1 (WIP1) also dephosphorylates ␥-H2AX at serine 139 in vitro and in vivo. Overexpression of WIP1 reduces formation of ␥-H2AX foci in response to ionizing and ultraviolet radiation and blocks recruitment of MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1) to DNA damage foci. Finally, these inhibitory effects of WIP1 on ␥-H2AX are accompanied by WIP1 suppression of DNA double strand break repair. Thus, WIP1 has a homeostatic role in reversing the effects of ataxia telangiectasia-mutated phosphorylation of H2AX.