Rad52-Independent Accumulation of Joint Circular Minichromosomes during S Phase in Saccharomyces cerevisiae

Abstract

We investigated the formation of X-shaped molecules consisting of joint circular minichromosomes (joint molecules) in Saccharomyces cerevisiae by two-dimensional neutral/neutral gel electrophoresis of psoralencross-linked DNA. The appearance of joint molecules was found to be replication dependent. The joint molecules had physical properties reminiscent of Holliday junctions or hemicatenanes, as monitored by strand displacement, branch migration, and nuclease digestion. Physical linkage of the joint molecules was detected along the entire length of the minichromosome and most likely involved newly replicated sister chromatids. Surprisingly, the formation of joint molecules was found to be independent of Rad52p as well as of other factors associated with a function in homologous recombination or in the resolution of stalled replication intermediates. These findings thus imply the existence of a nonrecombinational pathway(s) for the formation of joint molecules during the process of DNA replication or minichromosome segregation.DNA replication is a highly coordinated process providing an accurate duplication of the chromosomes required for faithful transmission of the genomic information from mother to daughter cells. Studies of mitotic cells have led to a unified model for DNA replication involving three distinct steps; initiation, elongation, and termination (for a review, see reference 57). Several studies seem to corroborate the general features of the bidirectional replication model, which proposes that pairs of divergent replication forks travel away from their origins until they meet the forks coming from opposite directions from the neighboring replicons (22, 42). When two replication forks converge at the end of DNA synthesis, the leading-end polymerases advance toward each other, melting the remaining duplex region, and finish the replication by producing catenated DNA molecules (50,51). An alternative model for termination proposed that one of the advancing polymerases dislodges from the template by accumulated torsional stress, so that only one fork can advance. In subsequent reaction steps, including strand displacement and branch migration, hemicatenated DNA is formed and resolved by type I topoisomerases (31).DNA recombination is an integral part of DNA replication and contributes to the structural integrity of the newly replicated genome (for recent reviews, see references 3, 10, and 30). In replicating DNA, X-shaped molecules (xDNA) with properties of Holliday junction intermediates of homologous recombination (21) can be distinguished physically from replicating molecules by two-dimensional gel electrophoresis (5, 43). The formation of such joint molecules was recently studied within the Saccharomyces cerevisiae ribosomal DNA (rDNA) locus (60) and various loci of Physarum polycephalum (6). In both organisms, Holliday junction-like xDNA molecules were more abundant in S phase. Interestingly, mutations in polymerases ␣ and ␦ stimulated the formation of Holliday junctions in the S. cerevisiae rDNA l...

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase

“…The abnormal accumulation of X molecules observed in MMS-treated sgs1 cells, together with the observation that a population of X structure is indeed able to migrate with the same kinetics as in MMS-treated wild-type cells, raises the question as to whether the X structures that keep accumulating at late time points represent SCJs or, rather, different intermediates. It should be noted that at least two types of molecules migrate like an X spike on 2D gels: hemicatenanes and HJs (Collins and Newlon 1994;Lockshon et al 1995;Schwacha and Kleckner 1995;Zou and Rothstein 1997;Lucas and Hyrien 2000;Lopes et al 2003;Wellinger et al 2003). Given that SCJs have been suggested to represent hemicatenanes (Lopes et al 2003), we tested whether the X molecules abnormally accumulating in MMS-treated sgs1 cells were instead recombination-dependent structures.…”

“…In general, four-branched molecules migrate on 2D gels in the so-called X spike (Brewer and Fangman 1987). X-shaped structures on 2D gels have been related to HJs (Collins and Newlon 1994;Lockshon et al 1995;Schwacha and Kleckner 1995;Zou and Rothstein 1997) or to hemicatenanes (Lucas and Hyrien 2000;Lopes et al 2003;Wellinger et al 2003). The X-shaped SCJ structure Figure 7.…”

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

“…In accordance with a replicon-like structure, the bubble-shaped molecules were resistant to in vitro RNase H treatment and heat-induced branch migration. Furthermore, as expected for replicating molecules having an extendable 3′ end, in vitro DNA synthesis by Klenow/gp32 treatment could destroy the bubble-shaped molecules by strand displacement (43). These analyses rule out the presence of long stretches of R-loops, which could have a bubble-like appearance, and suggest a rapid conversion of R-loops into noncanonical replicons.…”

Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex

“…After the culture was brought to 1% glucose or 1% galactose, cells were grown for another 3.5 to 4 h prior to DNA isolation. DNA isolation, restriction enzyme digestion, and two-dimensional (2D) gel analysis were performed as described previously (45). Prior to 2D gel analysis, the amount of plasmid DNA was determined to adjust for equal loadings.…”