Increased expression of Polδ 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 Polδ 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 Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics  in vivo  using the fission yeast  Schizosaccharomyces pombe  as a model system.  Methods:  To generate  S. pombe  strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of  cdc1  +   ,  cdc6  +   (or  cdc6  L591G   ),  cdc27  +   and  cdm1  +   by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ 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 Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ 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 Polε for Polδ does occur i  n vivo , it represents a rare phenomenon that does not significantly influence canonical replication program.
Zach R, Carr AM