Abstract
In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.
Highlights
Chromosomal DNA replication origins function as sites where enzymes assemble to form the replication fork machinery
Genetic Analysis of the N-terminal Region of DnaA—We investigated the function of 10 amino acid residues contained within an N-terminal region of DnaA, asking whether these residues are essential
The results indicate that L3A, F46A, and L62A were comparable or nearly comparable with wild type dnaA in supporting DNA replication of the oriC plasmid, whereas L10A was about 3-fold reduced in activity
Summary
Replication Proteins—Wild type DnaA protein fused at its N terminus to polyhistidine (His-Tag®; Novagen) was purified from an overproducing strain carrying pKC597 (see below) as described [17], but the step of heparin-agarose chromatography was substituted by metal chelation chromatography. Monomeric His-tagged DnaA is essentially identical to wild type DnaA in oriC plasmid replication in either a. The mutant DnaA carrying the W6A substitution was purified essentially as described above The His tag modification has been omitted from the protein nomenclature of wild type DnaA and W6A. DnaA⌬62, which lacks the N-terminal 62 amino acids of DnaA and does not carry a His tag, was purified as described [11]. DNA fragments carrying oriC or oriV from plasmid RK2 were prepared by cleavage of M13oriC2LB5 with EcoRI or of pSP6 with EcoRI and HincII endonucleases, respectively. After incubation with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (Bio-Rad), chemiluminescence (Supersignal; Pierce) was detected with x-ray film (X-Omat, Eastman Kodak Co.)
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