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PMID: 19968790 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo.

Molecular microbiology ·Vol. 75 ·No. 2 ·2010-01-00 ·Pages 452-61

Smits WK, Goranov AI, Grossman AD

Abstract

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.

MeSH Terms
Bacillus subtilis/enzymology,genetics Bacterial Proteins/genetics,metabolism Carrier Proteins/genetics,metabolism DNA Helicases/genetics,metabolism DNA Replication/genetics DNA-Binding Proteins/genetics,metabolism Kinetics Models, Genetic Mutagenesis Mutagenesis, Insertional Origin Recognition Complex/genetics Replication Origin Thermodynamics
Chemicals
Bacterial Proteins Carrier Proteins DNA-Binding Proteins DnaA protein, Bacteria DnaD protein, Bacillus subtilis OriC chromosomal replication origin Origin Recognition Complex DNA Helicases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Smits Wiep Klaas
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Goranov Alexi I
Grossman Alan D
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Article Info
Journal
Molecular microbiology
Abbr.
Mol Microbiol
ISSN
1365-2958
DOI
10.1111/j.1365-2958.2009.06999.x
Published
2010-01-00
Epub
2009-00-04
Pages
452-61
Language
English
Region
England
NLM ID
8712028
PMCID
PMC2992960
Subset
IM
Grants
NIGMS NIH HHS · R37 GM041934-18 · United States
NIGMS NIH HHS · R37 GM041934 · United States
NIGMS NIH HHS · R01 GM041934-17 · United States
NIGMS NIH HHS · R01 GM041934 · United States
NIGMS NIH HHS · GM41934 · United States
External Links
PubMed source DOI: 10.1111/j.1365-2958.2009.06999.x PMC Full Text View journal details
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