A hybrid bacterial replication origin

Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany.
EMBO Reports (Impact Factor: 9.06). 12/2001; 2(11):1003-6. DOI: 10.1093/embo-reports/kve225
Source: PubMed


We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

Download full-text


Available from: Harald Seitz, Mar 05, 2014
  • Source
    • "In M.tb, the DNA fragments bearing the dnaA-dnaN intergenic region function as oriC [5]. Upon comparison of the oriC region of E. coli, M.tb and B. subtilis (Figure 1A) it appears that E. coli has three A+T rich 13 mers [1], B. subtilis has a 27 mer [4] which is exclusively rich in A+T residues, but M.tb has only one A+T rich 15 mer region [5], [22]. It should also be noted that E. coli has only 5 DnaA boxes (Figure 1B) whereas M.tb has 13 such boxes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis (M.tb), the pathogen that causes tuberculosis, is capable of staying asymptomatically in a latent form, persisting for years in very low replicating state, before getting reactivated to cause active infection. It is therefore important to study M.tb chromosome replication, specifically its initiation and regulation. While the region between dnaA and dnaN gene is capable of autonomous replication, little is known about the interaction between DnaA initiator protein, oriC origin of replication sequences and their negative effectors of replication. By KMnO(4) mapping assays the sequences involved in open complex formation within oriC, mediated by M.tb DnaA protein, were mapped to position -500 to -518 with respect to the dnaN gene. Contrary to E. coli, the M.tb DnaA in the presence of non-hydrolysable analogue of ATP (ATPgammaS) was unable to participate in helix opening thereby pointing to the importance of ATP hydrolysis. Interestingly, ATPase activity in the presence of supercoiled template was higher than that observed for DnaA box alone. M.tb rRv1985c, a homologue of E.coli IciA (Inhibitor of chromosomal initiation) protein, could inhibit DnaA-mediated in-vitro helix opening by specifically binding to A+T rich region of oriC, provided the open complex formation had not initiated. rIciA could also inhibit in-vitro replication of plasmid carrying the M.tb origin of replication. These results have a bearing on the functional role of the important regulator of M.tb chromosomal replication belonging to the LysR family of bacterial regulatory proteins in the context of latency.
    Full-text · Article · Feb 2009 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: The plasmids pN42 and pJBL2 were isolated from the Lactobacillus delbrueckii subsp. lactis strains NCC88 and JCL414. DNA sequence determination and bioinformatic analysis revealed a strikingly conserved genetic organization containing five major, highly conserved open reading frames (ORFs). Transformation studies indicated that ORF2 (consisting of a primase fused to a replicative DNA helicase), ori, and ORF3 constitute the minimal requirements for replication of pN42 in the heterologous host Lactococcus lactis. The ORF1's are predicted to encode type I restriction-modification (R-M) system HsdS subunits with different specificities on either plasmid, suggesting that these plasmids may be involved in host defense by expanding their host R-M system repertoire. These plasmids constitute the basis for the construction of novel L. delbrueckii vectors.
    No preview · Article · Apr 2002 · Plasmid
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.
    Preview · Article · Dec 2002 · FEMS Microbiology Reviews
Show more