• Title/Summary/Keyword: Replication initiator protein

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The Bacteriophage λ DNA Replication Protein P Inhibits the oriC DNA- and ATP-binding Functions of the DNA Replication Initiator Protein DnaA of Escherichia coli

  • Datta, Indrani;Sau, Subrata;Sil, Alok Kumar;Mandal, Mitai C.
    • BMB Reports
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    • v.38 no.1
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    • pp.97-103
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    • 2005
  • Under the condition of expression of $\lambda$ P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the $\lambda$ P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the $\lambda$ P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of $\lambda$ P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

Molecular Interactions of a Replication Initiator Protein, RepA, with the Replication Origin of the Enterococcal Plasmid p703/5

  • Cha, Kyung-Il;Lim, Ki-Hong;Jang, Se-Hwan;Lim, Wang-Jin;Kim, Tae-Hyung;Chang, Hyo-Ihl
    • Journal of Microbiology and Biotechnology
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    • v.17 no.11
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    • pp.1841-1847
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    • 2007
  • We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteroncarrying theta-type plasmids.

Binding of IciA protein to the dnaA promoter region

  • Kim, Hakjung;Hwang, Deog-Su
    • Journal of Microbiology
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    • v.33 no.3
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    • pp.191-195
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    • 1995
  • IciA protein has been shown as an inhibitor for the initiation of E. coli chromosomal DNA replication at oriC. IciA protein binds the AT-rich region in oriC and then blocks the initiation of chromosomal DNA replication. Two binding sites for IciA protein were identified in dnaA gene, encoding the initiator for the E. coli chromosomal replication, promoter region by gel-shift assay and DNase I footprinting, One, named as IciA site I, is located upstream of the dnaA promoter 1P. The other, named as IciA site II, is located downstream of the dnaA promoter 2P. The sequence comparison of the regions protected from the DNase I cleavage did not result in a clear consensus sequence for the binding of IciA protein, suggesting that IciA protein may be a member of multimeric complex dsDNA binding proteins. This study provided information about the binding mode of IciA protein. Even though the IciA site II and IciA binding site in oriC seem to be composed of two IciA binding units, one binding unit is likely enough to cause the binding of IciA protein to the IciA site I. The binding of IciA protein to the dna4 promoter implies that IciA protein may involve not only the control of the initiation of chromosomal DNA replication but also the control of the dna4 gene expression.

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Characterization of the Small Cryptic Plasmid, pGD2, of Klebsiellia sp. KCL-2.

  • Yoo, Ju-Soon;Kim, Hae-Sun;Chung, Soo-Yeol;Lee, Young-Choon;Cho, Young-Soo;Choi, Yong-Lark
    • BMB Reports
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    • v.34 no.6
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    • pp.584-589
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    • 2001
  • One of the cryptic plasmids from the oil degrading bacterium Klebsiella sp. KCL-2, the small plasmid pGD2, has been identified and characterized. This plasmid has a size of 3.6 kb with unknown functions. We constructed the recombinant plasmid pMGD2. The nucleotide sequences of the plasmid were determined and two open reading frames were detected. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology with the protein of ColE2 plasmid. The product encoded by ORF2 showed a high similarity with the transposase protein of IS5. IS5 is 1195 by long and contains an inverted terminal repetition of 16 bp with one mismatch. Stem-loop structures in the 5'untranslated region of the repA suggest that a putative gene, incA, is located in a complementary strand to the leader region of the repA mRNA.

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Crystallization of Escherichia coli IciA Protein An Initiation of Chroirnsomal Replication (대장균 염색체 복제 개시 저해제, IciA 단백질의 결정화)

  • Song, Hyun-Kyu;Cha, Hoon;Yoo, Soon-Ji;Chung, Chin-Ha;Hwang, Deog-Su;Suh, Se-Won
    • Korean Journal of Crystallography
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    • v.5 no.1
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    • pp.20-23
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    • 1994
  • Specific binding to the oric region of E, coli chromsome by IciA protein inhibits initiation of chrorrnsomal replication in vitro by blocking the opening of this region effected by the initiator DnaA protein. The IciA protein has been suggested play a critical role in a key stage of the cell cycle. In order to study the structure-function relationship of IciA protein, we are determining the three-dimensional structure of IciA Votein by X-ray crystallography, As a first step toward its structure detumination E. coli IciA protein has been crystallized using sodium formate as a precipitant.

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Trigger Factor Interacts with DnaA Protein to Stimulate its Interaction with DnaA Box

  • Lee, Yong-Sun;Lee, June;Kim, Hak-Kyun;Kang, Sukhyun;Han, Joo-Seok;Kim, Jae-Bum;Hwang, Deog-Su
    • Animal cells and systems
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    • v.7 no.1
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    • pp.81-87
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    • 2003
  • While screening proteins that interact with DnaA protein, the initiator protein for Escherichia coil chromosomal DNA replication, we found a 52-kD sized protein which bound to DnaA protein in a salt-dependent manner. This protein was identified as trigger factor, a ribosome-associated peptidyl-prolyl- cisltrans isomerase with chaperone activity. Trigger factor was overproduced and purified to near homogeneity, and its effect on the function of DnaA protein was examined, Enhanced binding of DnaA protein to DnaA box with no apparent supershift in the gel-shift experiments suggested that trigger factor, by virtue of its chaperone activity, exerts a change on DnaA protein thus increasing its binding affinity for DnaA box.

pVC, a Small Cryptic Plasmid from the Environmental Isolate of Vibrio cholerae MP-1

  • Zhang, Ruifu;Wang, Yanling;Leung, Pak Chow;Gu, Ji-Dong
    • Journal of Microbiology
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    • v.45 no.3
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    • pp.193-198
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    • 2007
  • A marine bacterium was isolated from Mai Po Nature Reserve of Hong Kong and identified as Vibrio cholerae MP-1. It contains a small plasmid designated as pVC of 3.8 kb. Four open reading frames (ORFs) are identified on the plasmid, but none of them shows homology to any known protein. Database search indicated that a 440 bp fragment is 96% identical to a fragment found in a small plasmid of another V. cholerae. Further experiments demonstrated that a 2.3 kb EcoRI fragment containing the complete ORF1, partial ORF4 and their intergenic region could self-replicate. Additional analyses revealed that sequence upstream of ORF1 showed the features characteristic of theta type replicons. Protein encoded by ORF1 has two characteristic motifs existed in most replication initiator proteins (Rep): the leucine zipper (LZ) motif located at the N-terminal region and the alpha helix-turn-alpha helix motif (HTH) located at the C-terminal end. The results suggest that pVC replicates via the theta type mechanism and is likely a novel type of theta replicon.

The Mutation that Makes Escherichia coli Resistant to λ P Gene-mediated Host Lethality Is Located within the DNA Initiator Gene dnaA of the Bacterium

  • Datta, Indrani;Banik-Maiti, Sarbani;Adhikari, Lopa;Sau, Subrata;Das, Niranjan;Mandal, Nitai Chandra
    • BMB Reports
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    • v.38 no.1
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    • pp.89-96
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    • 2005
  • Earlier, we reported that the bacteriophage $\lambda$ P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this $\lambda$ P gene-mediated lethality. In this paper, we show that under the $\lambda$ P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94% linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from $\lambda$ P gene-mediated killing and complements E. coli dnaAts46 at $42^{\circ}C$. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to $\lambda$ P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.