Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology. Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence. Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs. Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs. We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination. Gene conversion tract lengths were examined in 80 recombinants. Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less. These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.

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Repetitive elements comprise nearly half of the human genome. Chromosomal rearrangements involving these elements occur in somatic and germline cells and are causative for many diseases. To begin to understand the molecular mechanisms leading to these rearrangements in mammalian cells, we developed an intron-based system to specifically induce chromosomal translocations at Alu elements, the most numerous family of repetitive elements in humans. With this system, we found that when double-strand breaks (DSBs) were introduced adjacent to identical Alu elements, translocations occurred at high frequency and predominantly arose from repair by the single-strand annealing (SSA) pathway (85%). With diverged Alu elements, translocation frequency was unaltered, yet pathway usage shifted such that nonhomologous end joining (NHEJ) predominated as the translocation pathway (93%). These results emphasize the fluidity of mammalian DSB repair pathway usage. The intron-based system is highly adaptable to addressing a number of issues regarding molecular mechanisms of genomic rearrangements in mammalian cells.

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Recurrent reciprocal translocations are present in many hematologic and mesenchymal malignancies. Because significant sequence homology is absent from translocation breakpoint junctions, nonhomologous end-joining (NHEJ) pathways of DNA repair are presumed to catalyze their formation. We developed translocation reporters for use in mammalian cells from which NHEJ events can be selected after precise chromosomal breakage. Translocations were efficiently recovered with these reporters using mouse cells, and their breakpoint junctions recapitulated findings from oncogenic translocations. Small deletions and microhomology were present in most junctions; insertions and more complex events also were observed. Thus, our reporters model features of oncogenic rearrangements in human cancer cells. A homologous sequence at a distance from the break site affected the translocation junction without substantially altering translocation frequency. Interestingly, in a direct comparison, the spectrum of translocation breakpoint junctions differed from junctions derived from repair at a single chromosomal break, providing mechanistic insight into translocation formation. IntroductionMany hematologic and mesenchymal malignancies harbor reciprocal translocations that develop as early and essential events in oncogenesis. [1][2][3] Translocations likely result from contemporaneous DNA double-strand breaks (DSBs) generated by either endogenous (eg, V(D)J recombinase 4 ) or exogenous (eg, topoisomerase II poisons 5 ) agents. Mammalian cells have multiple pathways to repair DSBs, including mechanisms that involve little or no sequence homology, collectively termed nonhomologous endjoining (NHEJ). 6 NHEJ is important for survival in response to clastogens and is critical for the repair of DSBs induced during V(D)J recombination. 6,7 Homology-directed repair, an essential pathway in mammalian cells, 8 does not appear to be involved in the overwhelming majority of cancer-associated translocations, as breakpoint junctions lack extensive homology, 1-3 and translocations involving this pathway are not recovered in model systems. [9][10][11][12] However, another homologous repair pathway, single-strand annealing (SSA), efficiently generates translocations in model systems. 9,10 Given that reciprocal translocations in human cancer cells appear to arise from NHEJ, we developed translocation reporters for use in mammalian cells in which significant sequence homology is absent from the DNA ends. Study design DNA manipulations, transfections, and translocation analysisTargeting vectors, polymerase chain reaction (PCR) conditions, and primers are described in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). For translocation experiments, 2 ϫ 10 7 cells were electroporated with 50 g pCBASce 9,13 or pCAGGS. 9,10 Fluorescence in situ hybridization (FISH) was performed as described. 9 Pool analysis of repair junctionsTwo p5rE cell lines and 2 translocation clones were electroporated with...

The correct repair of double-strand breaks (DSBs) is essential for the genomic integrity of a cell, as inappropriate repair can lead to chromosomal rearrangements such as translocations. In many hematologic cancers and sarcomas, translocations are the etiological factor in tumorigenesis, resulting in either the deregulation of a proto-oncogene or the expression of a fusion protein with transforming properties. Mammalian cells are able to repair DSBs by pathways involving homologous recombination and nonhomologous end-joining. The analysis of translocation breakpoints in a number of cancers and the development of model translocation systems are beginning to shed light on specific DSB repair pathway(s) responsible for the improper repair of broken chromosomes.

Rapidly growing cells of Saccharomyces cerevisiae are sensitive to heat shock, while non-growing stationary phase cells are highly resistant. We find that slowly growing cells have an intermediate degree of heat shock resistance that can be nearly as great as that of stationary phase cells. This resistance is correlated both with slow growth and with carbon catabolite derepression. Slowly growing cells also showed resistance to Zymolyase digestion of their cell walls. The stress resistance is a property of all the cells in the culture, and cell cycle position makes little difference to the degree of stress resistance. At least some of the properties normally associated with stationary phase cells do not require residence in stationary phase or any other particular compartment of the cell cycle. Stress resistance may be due to a diverse set of physiological adaptations available to cells regardless of their position in the cell cycle. That is, although stress resistance and stationary phase are often correlated, neither is the cause of the other.

We isolated a mutant strain unable to acquire heat shock resistance in stationary phase. Two mutations contributed to this phenotype. One mutation was at the TPS2locus, which encodes trehalose-6phosphate phosphatase. The mutant fails to make trehalose and accumulates trehalose-6-phosphate. The other mutation was at the HSP104 locus. Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells. The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance. The same effect was seen in the tps1 (trehalose-6phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6 phosphate. However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth. Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants. The encoded protein has a region homologous to the active site of phosphomutases.