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Rannou, Olivier and Le Chatelier, Emanuelle and Larson, Marilynn A. and Nouri, Hamid and Dalmais, Bérengère and Laughton, Charles and Laurent, Jannière and Soultanas, Panos (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). pp. 5303-5320. ISSN 0305-1048

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.

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