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- Title
DnaA-ATP-specific binding sites in E. coli oriC are required for correct timing of initiation of DNA replication.
- Authors
Rozgaja, Tania; Grimwade, Julia E.; Torgue, Julien J.-C.; Leonard, Alan C.
- Abstract
Initiation of DNA replication is precisely timed during the cell cycle to ensure that daughter cells receive equal genomes. In eukaryotes, archea, and eubacteria, initiation requires that structurally conserved ATP-binding initiator proteins interact with replication origins in pre-replication complexes (pre-RCs). In E. coli, initiator DnaA first forms an origin recognition complex (ORC) at high affinity binding sites in the chromosomal origin, oriC. Initiation is triggered by converting ORC to pre-RC when DnaA fills lower affinity sites. Two low affinity sites preferentially bind DnaA-ATP, and single base alterations in both sites eliminate this preference. To examine the role of DnaA-ATP sites in initiation timing, replication of mutated oriC plasmids was examined. Plasmids with oriC lacking discriminatory sites replicated early and more than once in the cell cycle, and had a higher copy number than wild-type origin plasmids. Mutant oriC-pBR322 plasmids exhibited severe incompatibility with wild type chromosomal oriC, resulting in spontaneous replacement of host oriC with mutant oriC. This incompatibility could be reversed by conversion of a non-discriminatory low affinity site into one that preferred DnaA-ATP. These data indicate that binding of DnaA-ATP to low affinity sites plays a role in initiation timing, and there is plasticity in the positioning of discriminatory sites in oriC, NIH GM54042.
- Subjects
BINDING sites; ESCHERICHIA coli; DNA replication; PROTEINS; PLASMIDS
- Publication
FASEB Journal, 2007, Vol 21, Issue 5, pA658
- ISSN
0892-6638
- Publication type
Article
- DOI
10.1096/fasebj.21.5.a658-c