Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo.
The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature-sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI-mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.
Smits
Wiep Klaas
WK
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Goranov
Alexi I
AI
Grossman
Alan D
AD
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D051738:Origin Recognition Complex / Q000235:genetics; D018741:Replication Origin; D013816:Thermodynamics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C092895:DnaD protein, Bacillus subtilis; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D004265:DNA Helicases