Rapid Analysis of Saccharomyces cerevisiae Genome Rearrangements by Multiplex Ligation–Dependent Probe Amplification

Abstract

Aneuploidy and gross chromosomal rearrangements (GCRs) can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation–dependent Probe Amplification (MLPA) to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1–Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109–dependent acetylation of histone H3 lysine residue 56 (H3K56) resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.

Author Summary

In this study we describe an adaptation of Multiplex Ligation–dependent Probe Amplification (MLPA) for use in the study of gross chromosomal rearrangements (GCRs) that occur in S. cerevisiae mutants with increased genome instability. Our previous study found that the presence of a Ty912 element on a nonessential arm of chromosome V resulted in increased rates of non-reciprocal monocentric translocations arising from recombination between the Ty912 on chromosome V and ectopic Ty elements on other chromosomes. Using MLPA, we observed that the majority of the translocations targeted six different translocation hotspots even though there were at least 254 potential targets for Ty-mediated translocations in the S. cerevisiae genome. Most of the observed translocations were formed by RAD52-dependent recombination, although we also identified a RAD52-independent recombination pathway that promoted the formation of the same types of translocations at lower rates. Finally, we found that defects in the ASF1-RTT109–dependent histone H3 lysine 56 (H3K56) acetylation pathway caused increased rates of both Ty-mediated translocations and whole-chromosome duplications (aneuploidy). This aneuploidy often occurred simultaneously with Ty-mediated translocations. Overall, our results demonstrate that MLPA is a rapid, inexpensive method that allows the analysis of the large number of GCRs needed to understand the pathways that suppress or promote genome instability.