Lack of chromosome territoriality in yeast: promiscuous rejoining of broken chromosome ends - PubMed (original) (raw)

Lack of chromosome territoriality in yeast: promiscuous rejoining of broken chromosome ends

J E Haber et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Various studies suggest that eukarytoic chromosomes may occupy distinct territories within the nucleus and that chromosomes are tethered to a nuclear matrix. These constraints might limit interchromosomal interactions. We have used a molecular genetic test to investigate whether the chromosomes of Saccharomyces cerevisiae exhibit such territoriality. A chromosomal double-strand break (DSB) can be efficiently repaired by recombination between flanking homologous repeated sequences. We have constructed a strain in which DSBs are delivered simultaneously to both chromosome III and chromosome V by induction of the HO endonuclease. The arrangement of partially duplicated HIS4 and URA3 sequences around each HO recognition site allows the repair of the two DSBs in two alternative ways: (i) the creation of two intrachromosomal deletions or (ii) the formation of a pair of reciprocal translocations. We show that reciprocal translocations are formed approximately as often as the pair of intrachromosomal deletions. Similar results were obtained when one of the target regions was moved from chromosome V to any of three different locations on chromosome XI. These results argue that the broken ends of mitotic chromosomes are free to search the entire genome for appropriate partners; thus, mitotic chromosomes are not functionally confined to isolated domains of the nucleus, at least when chromosomes are broken.

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Figures

Figure 1

Figure 1

Alternative ways of repairing two DSBs. In the nucleus of a haploid strain illustrated here, repair of two DSBs by SSA can occur in two different ways. There may be two intrachromosomal deletion events (A); alternatively, there may be a pair of reciprocal translocations by annealing between equivalently sized homologous regions on two different chromosomes (B).

Figure 2

Figure 2

Repair of two broken chromosomes by single-stand annealing between URA3 and_HIS4_ sequences on chromosomes III and V. (A) pBR322 plasmids carrying URA3 and Δ_his4_Δ sequences and the LEU2 gene flanking by a pair of HO cs were integrated into chromosome III (at_HIS4_) and chromosome V (at ura3-52), as shown. The ura3-52 allele contains a large insertion of a Ty element. After induction of HO endonuclease, the broken chromosomes may be repaired by two intrachromosomal deletion events, leading to the loss of all plasmid sequences (vertical hatched lines). Alternatively, there may be a pair of reciprocal translocations by annealing between equivalently sized homologous regions on two different chromosomes. The vertical dotted lines indicate the positions of _Pvu_II restriction endonuclease sites. (B) Southern blot analysis of the kinetics of appearance of deletions and translocations. DNA was extracted from cells at the intervals indicated after HO induction and digested with _Pvu_II. The Southern blot was probed with Δ_his4_Δ fragment indicated by the dark diagonally hatched lines in A. The fragments characteristic of a pair of intrachromosomal deletions (His+ Ura−) and of a pair of reciprocal translocations (His− Ura+) are shown on the right side of the figure. Two examples of each type are shown. cut* indicates the expected size of a Pvu_II restriction fragment if HO cleaves only the centromere-proximal site on chromosome III, thus replacing fragment “cut1” with a 6.4-kb fragment (see_A, above). The 4.7-kb fragment is present in both the parental strain and in derivatives containing reciprocal translocations. The 4.2-kb fragment is diagnostic of reciprocal translocations while the 2.4-kb fragment is indicative of an intrachromosomal deletion.

Figure 3

Figure 3

Repair of two broken chromosomes by single-stand annealing between URA3 and HIS4 sequences on chromosomes III and XI. (A) pBR322 plasmids pJH825 and pJH1113 carrying URA3 and Δ_his4_Δ sequences and the LEU2 gene flanking by a pair of HO cs were integrated into chromosome III (at HIS4) and chromosome XI (at YKL0086::URA3), respectively, as shown. After induction of HO endonuclease, the broken chromosomes may be repaired by two intrachromosomal deletion events (A), leading a His+ cell in which there has been a loss of all plasmid sequences (vertical hatched lines). Alternatively, there may be a pair of reciprocal translocations (B), producing a His− cell. Similar analyses were carried out where the chromosome XI target was at_apn1_::URA3 or_tif1_::URA3 (see below). (B) Locations of three sites where the_URA3_-pBR322-cs::LEU2::cs-URA3 target was inserted. The distances (in kb) indicate the position of each target relative to the left telomere. The frequencies of reciprocal translocations between the target and the site on chromosome III were scored as His− Ura+ cells (see above) and are indicated for each construct.

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