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, B�reng�re; Laughton, Charles; Laurent, Janni�re; Soultanas, Panos

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.

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), https://doi.org/10.1093/nar/gkt207

• Journal Article Type: Article
• Publication Date: May 1, 2013
• Deposit Date: Jul 15, 2013
• Publicly Available Date: Jul 15, 2013
• Journal: Nucleic Acids Research
• Print ISSN: 0305-1048
• Electronic ISSN: 1362-4962
• Publisher: Oxford University Press
• Peer Reviewed: Peer Reviewed
• Volume: 41
• Issue: 10
• DOI: https://doi.org/10.1093/nar/gkt207
• Public URL: https://nottingham-repository.worktribe.com/output/714075
• Publisher URL: http://www.ncbi.nlm.nih.gov/pubmed/23563155