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Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

Rannou, Olivier; Le Chatelier, Emanuelle; Larson, Marilynn A.; Nouri, Hamid; Dalmais, Bérengère; Laughton, Charles; Laurent, Jannière; Soultanas, Panos

Authors

Olivier Rannou

Emanuelle Le Chatelier

Marilynn A. Larson

Hamid Nouri

Bérengère Dalmais

Charles Laughton

Jannière Laurent

Panos Soultanas panos.soultanas@nottingham.ac.uk

Abstract

Bacillus subtilis has two replicative DNA polymerases.
PolC is a processive high-fidelity replicative
polymerase, while the error-prone DnaEBs extends
RNA primers before hand-off to PolC at the lagging
strand. We show that DnaEBs interacts with the replicative
helicase DnaC and primase DnaG in a
ternary complex. We characterize their activities
and analyse the functional significance of their interactions
using primase, helicase and primer extension
assays, and a ‘stripped down’ reconstituted
coupled assay to investigate the coordinated displacement
of the parental duplex DNA at a replication
fork, synthesis of RNA primers along the
lagging strand and hand-off to DnaEBs. The DnaG–
DnaEBs hand-off takes place after de novo polymerization
of only two ribonucleotides by DnaG, and
does not require other replication proteins.
Furthermore, the fidelity of DnaEBs is improved by
DnaC and DnaG, likely via allosteric effects induced
by direct protein–protein interactions that lower the
efficiency of nucleotide mis-incorporations and/or
the efficiency of extension of mis-aligned primers
in the catalytic site of DnaEBs. We conclude that
de novo RNA primer synthesis by DnaG and initial
primer extension by DnaEBs are carried out by a
lagging strand–specific subcomplex comprising
DnaG, DnaEBs and DnaC, which stimulates chromosomal
replication with enhanced fidelity.

Journal Article Type Article
Publication Date May 1, 2013
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 0305-1048
Publisher Oxford University Press
Peer Reviewed Peer Reviewed
Volume 41
Issue 10
Institution Citation Rannou, O., Le Chatelier, E., Larson, M. A., Nouri, H., Dalmais, B., Laughton, C., …Soultanas, P. (2013). Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis. Nucleic Acids Research, 41(10), doi:10.1093/nar/gkt207
DOI https://doi.org/10.1093/nar/gkt207
Publisher URL http://www.ncbi.nlm.nih.gov/pubmed/23563155
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0




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