Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication.
Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.
Bruand
Claude
C
Laboratoire de Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas, France.
Velten
Marion
M
McGovern
Stephen
S
Marsin
Stéphanie
S
Sérèna
Céline
C
Ehrlich
S Dusko
SD
Polard
Patrice
P
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000235:genetics; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases; D007700:Kinetics; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C092895:DnaD protein, Bacillus subtilis; D011994:Recombinant Proteins; D004265:DNA Helicases; D053488:DnaB Helicases