• 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.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.4
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• pp.138-142
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• 2016

Replication Protein A (RPA) is the eukaryotic single-stranded DNA binding protein. It involves in DNA replication, repair, and damage response. Among three subunits, RPA70 has a protein-protein binding domain (RPA70N) at the N-terminal. It has known that the domain recruits several damage response proteins to the damaged site. Also, it is suggested that there are more candidates that interact with RPA70N. Even though several studies performed on the structural aspects of RPA70N and its ligand binding, the backbone assignments of RPA70N is not available in public. In this study, we present the backbone assignments of RPA70N.

• 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.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• Korean Journal of Microbiology
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• v.34 no.4
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• pp.248-253
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• 1998

Bacteriophage N4, a lytic phage specific for Esherichia coli K12 strain encodes single-stranded DNA-binding protein, N4SSB (bacteriophage N4-coded single-stranded DNA-binding protein). N4SSB protein is originally identified as a protein required for N4 DNA replication. N4SSB protein is also required for N4 late transcription, which is catalyzed by E. coli ${\sigma}^{70}$ RNA polymerase. N4 late transcription does not occur until N4SSB protein is synthesized. Recently it is reported that N4SSB protein is essential for N4 DNA recombination. Therefore N 4SSB protein is a multifunctional protein required for N4 DNA replication, late transcription, and N4 DNA recombination. In this study, a variety of mutant N4SSB proteins containing internal deletions or substitutions were constructed to define and characterize domains important for N4 DNA replication, late transcription, and N4 DNA recombination. Test for the ill vivo activity of these mutant N4SSBs for N4 DNA replication, late transcription, and N4 DNA recombination was examined. The results suggest that C-terminal 7 amino acid residues are important for the activity of N4SSB. Three lysine residues, which are contained in this region play important roles on N4SSB activity.

• Molecules and Cells
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• v.44 no.2
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• pp.101-115
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• 2021

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.

• The Plant Pathology Journal
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• v.17 no.3
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• pp.115-122
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• 2001

Potato virus X (PVX) is a flexuous rod-shaped virus containing a single plus-strand RNA. Viral RNA synthesis is precisely regulated by regulatory viral sequences and by viral and/or host proteins. RNA sequence element as well as stable RNA stem-loop structure in the 5' end of the genome affect accumulation of genomic RNA and subgenomic RNA (sgRNA). The putative sgRNA promoter regions upstream of the PVX triple gene block (TB) and coat protein (CP) gene were critical for both TB and CP sgRNA accumulation. Mutations that disrupted complementarity between a region at the 5' end of the genomic RNA and the sequences located upstream of each sgRNA initiation site is important for PVX RNA accumulation. Compensatory mutations that restore complementarity restored sgRNA accumulation levels. However, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Gel-retardation assays showed that the 5' end of the positive-strand RNA formed an RNA-protein complex with cellular proteins, suggesting possible involvement of cellular proteins for PVX replication. Future studies on cellular protein binding to the PVX RNA and their role in virus replication will bring a fresh understanding of PVX RNA replication.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.837-842
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• 2013

A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.

• BMB Reports
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• v.33 no.1
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• pp.59-62
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• 2000

Hepatitis C virus (HCV) nonstructural 5B (NS5B) protein is an RNA-dependent RNA polymerase (RdRp). To determine whether it can contribute to viral replication by interaction with cellular proteins, the yeast two-hybrid screening system was employed to screen a human liver cDNA library. Using the HCV NS5B as a bait, we have isolated positive clones encoding a cellular protein. The NS5B interacting protein, 5BIP, is a novel cellular protein of 170 amino acids. Interaction of the HCV NS5B protein with 5BIP was confirmed by a protein-protein blotting assay. Recently, we have demonstrated that NS5B possesses an RdRp activity and thus it is possible that 5BIP, in association with NS5B, plays a role in HCV replication.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.33.1-33.15
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• 2020

Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca²⁺-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.