Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis.
The regulation of initiation of DNA replication is crucial to ensure that the genome is replicated only once per cell cycle. In the Gram-positive bacterium Bacillus subtilis, the function of the YabA protein in initiation control was assigned based on its interaction with the DnaA initiator and the DnaN sliding clamp in the yeast two-hybrid and on the overinitiation phenotype observed in a yabA null strain. However, YabA is unrelated to known regulators of initiation and interacts with several additional proteins that could also be involved directly or not in initiation control. Here, we investigated the specific role of YabA interactions with DnaA and DnaN in initiation control by identifying single amino acid changes in YabA that disrupted solely the interaction with DnaA or DnaN. These disruptive mutations delineated specific interacting surfaces involving a Zn2+-cluster structure in YabA. In B. subtilis, these YabA interaction mutations abolished both initiation control and the formation of YabA foci at the replication factory. Upon coexpression of deficient YabA mutants, mixed oligomers formed foci at the replisome and restored initiation control, indicating that YabA acts within a heterocomplex with DnaA and DnaN. In agreement, purified YabA oligomerized and formed complexes with DnaA and DnaN. These findings underscore the functional association of YabA with the replication machinery, indicating that YabA regulates initiation through coupling with the elongation of replication.
Noirot-Gros
Marie-Françoise
MF
Laboratoire de Génétique Microbienne, Domaine de Vilvert, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France. marie-francoise.gros@jouy.inra.fr
Velten
M
M
Yoshimura
M
M
McGovern
S
S
Morimoto
T
T
Ehrlich
S D
SD
Ogasawara
N
N
Polard
P
P
Noirot
Philippe
P
D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D017354:Point Mutation; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase; C083980:dnaN protein, Bacteria