Formation of Complex and Unstable Chromosomal Translocations in Yeast

Abstract

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers.

Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability

“…Besides BIR, evidence of homology-length dependency is also seen in gene conversion, with Rad59 becoming increasingly important as the length of sequence homology decreases [59]. This length-dependency may also explain our observation that CAN1 / LYP1 / ALP1 rearrangements, which show short regions of homology at the breakpoints [10,60], are inhibited by deletion of RAD59 , but not by deletion of RAD51 . Despite this differential effect on chromosome rearrangements between CAN1 , LYP1 and ALP1 , we observed no difference in the rate of overall genome instability between sgs1Δ mec3Δ rad51Δ and sgs1Δ mec3Δ rad59Δ mutants (P = 0.6892, Table 2), suggesting that the DNA lesions that give rise to viable GCRs are accessible to multiple repair pathways.…”

“…95% confidence intervals (CIs) for median GCR rates were calculated according to Nair [80], where non-overlapping confidence intervals indicate statistically significant differences between median GCR rates. GCR rates for wildtype [81], sgs1 [82], mec3, sgs1 mec3 [60], rad51, sgs1 mec3 rad51 and sgs1 mec3 rad52 mutants [10] were reported previously and are included for comparison. b Rate of accumulating translocations between CAN1, LYP1 and/or ALP1 (Can r 5-FOA r × 10 -10) . GCR clones from sgs1, mec3, sgs1 mec3, sgs1 mec3 rad51, sgs1 mec3 rad52 were previously screened for CAN1/LYP1/ALP1 translocations [10,60].…”

“…GCR rates for wildtype [81], sgs1 [82], mec3, sgs1 mec3 [60], rad51, sgs1 mec3 rad51 and sgs1 mec3 rad52 mutants [10] were reported previously and are included for comparison.…”

Multiple cellular mechanisms prevent chromosomal rearrangements involving repetitive DNA

“…GCRs are more destructive than other types of genomic alterations because they can disrupt multiple genes at once and alter chromosome organization, making cells more susceptible to DNA damage (Stankiewicz and Lupski, 2002). Furthermore, a chromosome that receives a large genomic alteration is often less stable, either due to altered chromatin status or as in the case of a dicentric chromosome, prone to further rearrangement (Schmidt et al, 2010; Hastings et al, 2009; Fournier et al, 2010). Multiple breakages and fusions of unstable chromosomes are thought to contribute to chromothripsis, the rapid bursts of large-scale genomic rearrangements that can be found in many cancerous tumors (Stephens et al, 2011).…”

Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae

“…Basic amino acids are incorporated via three similar permeases, Can1, Lyp1, and Alp1, which share 60-65% sequence identity with each other 10. Can1 was originally identified as an arginine permease 11, though it has been also reported to transport lysine, histidine, and ornithine with lower affinities 12.…”