Stabilization of Dicentric Translocations through Secondary Rearrangements Mediated by Multiple Mechanisms in S. cerevisiae

Abstract

BackgroundThe gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instabilityMethodology/Principal FindingsThe structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.Conclusions/SignificanceHR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.

Cytokinesis breaks dicentric chromosomes preferentially at pericentromeric regions and telomere fusions

“…Fusion of the broken ends can create new dicentrics that break again in the following mitoses, engaging the cell lineage in successive breakage-fusion-bridge cycles (McClintock 1941). Within only a few cell divisions, this can result in large gene copy number aberrations through amplification and deletion of chromosomal regions (Ciullo et al 2002;Sabatier et al 2005;Stew enius et al 2005;Selvarajah et al 2006;Pennaneach and Kolodner 2009;Marotta et al 2013). It can also lead to chromothripsis (Sorzano et al 2013;Li et al 2014).…”

“…One is centromere inactivation through DNA rearrangements or epigenetic switches at one centromere (Lejeune et al 1973;Avarello et al 1992;Kramer et al 1994;Pennaneach and Kolodner 2009;Mackinnon and Campbell 2011;Sato et al 2012;Song et al 2013). The other is telomere addition at the broken ends; for instance, by telomerase or break-induced replication (Murnane and Sabatier 2004;Pennaneach and Kolodner 2009).…”

“…The survivors that emerge often display a deletion of one centromere (Mann and Davis 1983;Kramer et al 1994;Pennaneach and Kolodner 2009). These deletions require the nonhomologous end-joining double-strand break repair pathway, indicating that dicentrics are sometimes severed at centromeres (Kramer et al 1994;Lee et al 1999).…”

Dicentric breakage at telomere fusions

“…Most commonly studied dicentrics are created by double-strand break-induced rearrangements and were first described by Barbara McClintock in the 1940s (McClintock 1941(McClintock , 1942. They form anaphase bridges that break somewhere between the two centromeres, causing further rearrangements and genome instability, which eventually lead to cell death (Bajer 1964;Mann and Davis 1983;Haber et al 1984;Surosky and Tye 1985;Koshland et al 1987;Hill and Bloom 1989;Kramer et al 1994;Jannink et al 1996;Lo et al 2002;Han et al 2009;Paek et al 2009;Pennaneach and Kolodner 2009). The mechanism of dicentric breakage is unknown.…”

Bioinformatic identification of genes suppressing genome instability

“…A genetic assay developed in the yeast Saccharomyces cerevisiae has been used to identify genes and pathways that suppress gross chromosomal rearrangements (GCRs) mediated by single-copy DNA sequences (7). In this assay, selection against two genetic markers, CAN1 and URA3, placed on a nonessential end of the left arm of chromosome V selects for the loss of these two genes that results as a consequence of the formation of GCRs that delete the left arm of chromosome V. The types of GCRs that have been observed with this assay include terminal deletions healed by de novo telomere addition, translocations, isoduplications and other types of dicentric translocation chromosomes, interstitial deletions, circular chromosomes, and complex GCRs resulting from multiple cycles of rearrangement, usually as a result of the formation of unstable dicentric translocations (8)(9)(10)(11). Using this assay, oxidative defense pathways, the replication machinery, DNA repair pathways, cell cycle checkpoint pathways, telomere maintenance pathways, and chromatin modification and assembly pathways have been shown to function in concert to prevent genome rearrangements (reviewed in 12).…”