Chromosome breakage and repair.

FROM time to time one of my colleagues working at a medical school commiserates with me because I spend ∼40 hr a year lecturing to undergraduates. I always reply that teaching has compelled me to learn a lot of material that I would not have known about had I taught only my specialized subject. These forays into the “beyond” were instrumental in moving my research in new directions. Three articles that I published in Genetics in the early 1980s (McCusker and Haber 1981; Haber and Thorburn 1984; Haber et al. 1984) were the result of learning about, in order to teach, classic genetic experiments in Drosophila and maize. When I arrived at Brandeis I was assigned to teach genetics, a subject I had never studied as either an undergraduate or a graduate student2. I was fortunate to team up in teaching with Jeff Hall, a master geneticist, who taught me much of the lore of Drosophila and maize, to add to the yeast genetics that my lab and I were slowly learning3. I was particularly interested in Barbara McClintock's study of the “Activator (Ac)/Dissociator (Ds)” transposable elements whose excisions led to cycles of breakage–fusion–bridge (BFB) of broken chromosome ends that generated chromosomal truncations. Her studies resonated strongly with the behavior of apparently broken chromosomes that we were studying in budding yeast as a consequence of mating-type gene switching. The study of yeast mating-type (MAT) gene switching has provided gainful employment for many scientists interested in cell-type regulation, gene silencing, chromosome architecture, and DNA repair. Haploid cells express either the MATa or the MATα allele and mate with cells of the opposite type, but cells expressing both MATa and MATα are nonmating. But most unusual was that homothallic MATa cells could switch to MATα, or vice versa, as often as every cell division. Takano and Oshima (1970; Oshima and Takano 1971) showed that switching depended on two distant loci that they viewed as “controlling elements,” similar to those defined by McClintock in maize. Hicks et al. (1977) made the insightful suggestion that these two loci were in fact unexpressed copies of mating-type information (now called HMLα and HMRa) that could be transposed to replace the original MAT allele. Extending the mutational analysis of Mackay and Manney (1974) and the sometimes-published work of the inventive Don Hawthorne (Hawthorne 1963; see also Herskowitz 1988), Strathern et al. (1981) proposed that MATα encoded both a repressor of a-specific genes (MATα2) and a positive regulator of α-specific genes (MATα1). Thus a matα1 matα2 mutant proved to be a-like. The transposition/replacement of MAT alleles occurs very frequently in homothallic cells, expressing the HO gene encoding a site-specific endonuclease, but is very rare in heterothallic strains where HO is inactive. One way we tried to study these rare events was by mating two heterothallic MATα strains together, on the assumption that if one of them switched to MATa, it would readily conjugate with a MATα cell, as first shown by Hawthorne (1963). We took up this approach (McCusker and Haber 1981) and found that indeed ∼25% of MATα × MATα matings did appear to result from such switches, leading to stable MATa/MATα diploids. However, most of the events were different and were often genetically unstable, giving rise to colonies with multiple phenotypes. Many matings arose from the creation of an at least transient a-like cell (presumably lacking expression of both MATα1 and MATα2), allowing it to mate with another α-mater. In some cases the resulting diploids were 2n-1 aneuploids that had lost the entire, presumably broken, chromosome. In many instances the diploids that were still heterozygous for markers distal to the MAT locus, but now α-mating. The most interesting group resulted in the loss of the all the markers distal to and including MATα. Many of these colonies gave evidence of continuing genomic instability, with the loss of additional markers on chromosome III. Among nine stable derivatives we analyzed in detail, eight were homozygous for MATα and more distal markers, but one contained a truncation of the right arm from at least MAT to the end of the chromosome. This last type was reminiscent of outcomes described in maize after the transposition of the Ds element. The truncation in yeast was one of the first examples of the apparent acquisition of a new telomere, either by de novo addition or by a nonreciprocal translocation. Before the days of genome sequencing, it was not possible to be more precise. Viewed from today's perspective, we imagine that the diploids still heterozygous for the right arm repaired the chromosome break by gene conversion, becoming MATα/MATα. Those that became homozygous for the distal region likely resulted from break-induced replication (BIR, also known as recombination-dependent DNA replication) in which one end of the double-strand break (DSB) established a replication fork that could copy >100 kb to the end of the homologous, template chromosome (Morrow et al. 1997; Kraus et al. 2001; Davis and Symington 2004; Malkova et al. 2005). It is also possible that some of the diploids showing loss of heterozygosity for markers distal to MAT arose from reciprocal exchanges accompanying gene conversion because we did not recover instances in which the distal regions became homozygous wild type. In the same year that John McCusker examined MATα × MATα matings in heterothallic strains, Barbara Weiffenbach was studying similar genomic instability in homothallic strains (Weiffenbach and Haber 1981). Malone and Esposito (1980) had shown that MAT switching was lethal in the absence of the RAD52 recombination gene, suggesting that the process had similarities to the repair of X-ray-induced damage. It was not until 1982 that Strathern et al. (1982) showed that MAT switching did indeed involve a DSB. I had isolated a recessive mutation, swi1-1, that reduced, but did not eliminate, MAT switching (Garvik and Haber 1977); hence we could study a population of MATα rad52 cells in which several percent of the cells in each generation attempted to switch; such cells became a-like. This made it possible for us to examine newly generated broken chromosomes, importantly with all the breaks initiating at the same place. We mated these cells with a heterothallic MATα strain and recovered diploids in which the broken chromosome was repaired by gene conversion, by BIR, or by new telomere addition. We again found that many of these diploids exhibited genomic instability, so that the colonies that arose were sectored for markers on chromosome III. Again, there were cases where the broken chromosome was completely lost and others in which there were stable outcomes in which markers distal to MAT were hemizyous or homozygous4.