Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae

Abstract

IntroductionThe study of DNA repair mechanisms has been aided by the development of experimental systems that generate DNA double-strand breaks (DSBs) at specific chromosomal locations and during defined periods of the cell cycle. One such system is the budding yeast conditional dicentric chromosome (Hill and Bloom, 1989). This inducible dicentric chromosome was constructed by inserting a second copy of centromere 3 (CEN3) into chromosome III, approximately 45 kb upstream of the endogenous centromere 3 (Fig. 1A). The inserted centromere is under control of the GAL1 promoter, allowing for the reversible inactivation of this centromere when cells are grown in the presence of galactose. Studies utilizing dicentric chromosomes are unique in that DSBs are generated by mitotic pulling forces when centromeres on the same sister chromatid are aligned to opposite spindle poles during mitosis. Previous work showed that efficient healing of a broken dicentric chromosome in Saccharomyces cerevisiae requires RAD52, indicating that repair occurs primarily through homologous recombination . In cells lacking RAD52, infrequent dicentric chromosome repair events occur through non-homologous end joining (Kramer et al., 1994).Resolution of a conditional dicentric chromosome is accompanied by the deletion of one centromere and the intervening DNA. These events were initially thought to result from gene conversion (GC) accompanied by crossover (Jager and Philippsen, 1989). However, other studies of direct repeat recombination have shown that deletions commonly occur without generating the circular product predicted to form through a crossover event (Fishman-Lobell et al., 1992). Moreover, many deletion events are dependent on RAD1 and RAD10 (Klein, 1988;Schiestl and Prakash, 1990;Ivanov and Haber, 1995). The Rad1 and Rad10 proteins form a structurespecific endonuclease that generates 5′ single-stranded ends (Siede et al., 1993). These genes function in a recombination pathway known as single-strand annealing (SSA) (Sugawara and Haber, 1992;Sung et al., 1993;Ivanov and Haber, 1995). The recognition of SSA as a major pathway for direct repeat recombination suggests that this mechanism plays a role in the repair of a dicentric chromosome.Previous studies have shown that recombination frequency is primarily governed by the degree of sequence homology rather than the location of the recombining sequences within the genome (Haber and Leung, 1996). This implies the existence of a homology search mechanism as part of the recombination process. The integration of E. coli lacO and tetO sequences into chromosomes of yeast strains encoding lac repressor-GFP and tet repressor-GFP fusion proteins has recently provided visual evidence of long-distance interactions between like sequences (Aragon- Alcaide and Strunnikov, 2000). Such interactions may represent a constitutive homology search process. The mechanisms that bring homologous sequences into close association are unknown. Efficient meiotic recombination in the fission yeast Schizosaccharomyce...

Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes

“…It is possible that, in addition to passive foundational support, microtubules might be involved in the dynamic positioning of specific DNA regions. For example, homologous recombination, which requires two DNA loci to be spatially adjacent, is facilitated by microtubules in S. cerevisiae (Thrower et al 2003). While preliminary investigations of microtubules interacting directly with pol III transcription units in yeast have proven inconclusive (R.A. Haeusler and D.R.…”

“…The transcription-dependent tethering of some genes at the nuclear pore depends on interactions between the gene promoters and the basket of the nuclear pore complex (Schmid et al 2006). Centromere clustering at the spindle pole body requires microtubules (Bystricky et al 2004), as does homologous recombination, which requires spatial proximity of the two loci (Thrower et al 2003).…”

The role of homologous recombination repair in the formation of chromosome aberrations

“…However, wild-type mammalian cells after ionising radiation show a random interaction of chromosomes with no preference for homologue interactions (Cornforth et al, 2002). Recently it has been shown that recombination between direct repeats (SSA) in yeast requires functional microtubules, suggesting that microtubule-dependent chromosomal movement may be required (Thrower et al, 2003). However, in mammalian systems it is possible that interactions between repeat sequences on heterologous chromosomes will occur predominantly between neighbouring loci and the sufficiently large number of repeats in the mammalian genome may prevent the need for a large scale movement to search for a homologous sequence.…”

The 19S proteasome subunit Rpt3 regulates distribution of CENP-A by associating with centromeric chromatin

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