Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.
Bottom Line: We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion.The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing.Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.
Affiliation: IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.Show MeSH
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Mentions: Error-free DDT by TS can be molecularly monitored by examining the formation of X-shaped structures composed of sister chromatid junctions (SCJs) in the proximity of replication forks using 2D gel electrophoresis (Branzei et al., 2008). In this assay, yeast cells are released synchronously and allowed to replicate in media containing the alkylating reagent methyl methanesulfonate (MMS). The pattern of replication intermediates at genomic locations of interest is analyzed at different time points during DNA replication. Previous results showed that TS intermediates form during replication of damaged templates and accumulate when the Sgs1-Top3-Rmi1 complex is defective because of compromised resolution (Branzei et al., 2008; Liberi et al., 2005; Giannattasio et al., 2014). As a consequence, replication of damage templates leads to a progressive accumulation of SCJs in sgs1Δ mutant cells (Liberi et al., 2005), as well as in sgs1 hypomorphic mutants disrupted only in the helicase domain (Onoda et al., 2000; Vanoli et al., 2010) (Figure 1A).
Affiliation: IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.