Academic journal article Genetics

Defective Break-Induced Replication Leads to Half-Crossovers in Saccharomyces Cerevisiae

Academic journal article Genetics

Defective Break-Induced Replication Leads to Half-Crossovers in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Break-induced replication (BIR) is an important process of DNA metabolism that has been implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities, including loss of heterozygosity, translocations, and alternative telomere lengthening. Therefore, knowledge of how BIR is carried out and regulated is important for better understanding the maintenance of genomic stability in eukaryotes. Here we present a new yeast experimental system that enables the genetic control of BIR to be investigated. Analysis of mutations selected on the basis of their sensitivity to various DNA-damaging agents demonstrated that deletion of POL32, which encodes a third, nonessential subunit of polymerase δ, significantly reduced the efficiency of BIR, although some POL32-independent BIR was still observed. Importantly, the BIR defect in pol32Δ cells was associated with the formation of half-crossovers. We propose that these half-crossovers resulted from aberrant processing of BIR intermediates. Furthermore, we suggest that the half-crossovers observed in our system are analogous to nonreciprocal translocations (NRTs) described in mammalian tumor cells and, thus, our system could represent an opportunity to further study the NRT mechanism in yeast.

DOUBLE-strand DNA breaks (DSBs) often cause genetic instability due to the loss of important genetic information and, therefore, DSBs can threaten an organism's homeostasis. DSB-induced changes to the genome are implicated in a variety of human diseases, including birth defects and cancer. Thus, identification and characterization of the molecular mechanisms that repair DSBs are crucial for understanding how the integrity of living cells is maintained. Several different pathways to repair DSBs have been identified. In yeast, gene conversion (GC) is the preferred pathway to repair DSBs generated by endonucleases, ionizing radiation, or mechanical rupture of chromosomes. Several features of GC make it a "safe" pathway for DSB repair. First, GC proceeds via invasion of the two broken DNA ends into a homologous template, which ensures that the donor DNA is homologous to the recipient on both sides of the break. Second, the length of newly synthesized DNA is relatively short because it is limited to a short patch between the sites of invasion. Finally, in vegetative cells, GC is rarely associated with crossing over, which can lead to chromosomal rearrangements (Paques and Haber 1999; Ira et al. 2003).

Another pathway to repair DSBs is break-induced replication (BIR). According to existing models, BIR proceeds by invasion of one broken DNA end into the intact donor molecule, followed by initiation of DNA synthesis that can continue as far as the end of the donor chromosome (McEachern and Haber 2006). BIR can be dangerous for a cell because it can result in the copying of hundreds of kilobases of DNA from the donor molecule, while a large piece of the unrepaired, broken DNA can be lost. In addition, BIR can be initiated through strand invasion at ectopic chromosomal locations, which leads to chromosomal rearrangements, primarily translocations (Bosco and Haber 1998).

BIR was originally studied in bacteria, Escherichia coli, and in bacteriophage T4, where it is called replicationdependent repair (RDR) (Kogoma 1997; Kuzminov 1999; Kreuzer 2000; Marians 2000; Michel et al. 2001). In both of these organisms, BIR was shown to be involved in DSB repair. Several studies demonstrated that BIR operates in yeast, Saccharomyces cerevisiae (Voelkel-Meiman and Roeder 1990; Morrow et al. 1997; Bosco and Haber 1998; Davis and Symington 2004; Malkova et al. 2005). It has been shown that BIR is initiated during transformation of yeast with linearized DNA fragments (Morrow et al. 1997; Davis and Symington 2004). Also, BIR was implicated in the generation of nonreciprocal translocations following DSB induction with HO endonuclease (Bosco and Haber 1998; Lydeard et al. …

Search by... Author
Show... All Results Primary Sources Peer-reviewed

Oops!

An unknown error has occurred. Please click the button below to reload the page. If the problem persists, please try again in a little while.