Increased expression of Pold does not alter the canonical replication program in vivo

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Pold concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Pold represented a consequence of a relatively rare event, during which Pold stochastically outcompeted Pole and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Pold levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Pold, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Pold genes. To estimate expression of respective Pold genes in Pold-overexpressing mutants, we measured relative transcript levels of cdc1+, cdc6+ (or cdc6L591G), cdc27+ and cdm1+ by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Pold over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Pold does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Pold expression does not change activities of Pold, Pole and Pola at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Pold preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Pole for Pold does occur in vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.