Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved]
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 synthesi...
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doaj-25e2c150cc7246a9b3a9ee670ca46a9a2021-05-11T12:23:48ZengWellcomeWellcome Open Research2398-502X2021-05-01610.12688/wellcomeopenres.16600.218586Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved]Róbert Zach0Antony M. Carr1Genome Damage and Stability Centre, School of Life Sciences, Science Park Road, University of Sussex, Falmer, Brighton, BN1 9RQ, UKGenome Damage and Stability Centre, School of Life Sciences, Science Park Road, University of Sussex, Falmer, Brighton, BN1 9RQ, UKBackground: 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 cdc6L591G), 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 in vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.https://wellcomeopenresearch.org/articles/6-44/v2 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Róbert Zach Antony M. Carr |
spellingShingle |
Róbert Zach Antony M. Carr Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] Wellcome Open Research |
author_facet |
Róbert Zach Antony M. Carr |
author_sort |
Róbert Zach |
title |
Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
title_short |
Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
title_full |
Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
title_fullStr |
Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
title_full_unstemmed |
Increased expression of Polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
title_sort |
increased expression of polδ does not alter the canonical replication program in vivo [version 2; peer review: 2 approved] |
publisher |
Wellcome |
series |
Wellcome Open Research |
issn |
2398-502X |
publishDate |
2021-05-01 |
description |
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 cdc6L591G), 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 in vivo, it represents a rare phenomenon that does not significantly influence canonical replication program. |
url |
https://wellcomeopenresearch.org/articles/6-44/v2 |
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