title: Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1.
abstract: The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Sam Woong
authors.initials: SW
authors.lastname: Kim
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Jeong
authors.forename: Han Soo
authors.initials: HS
authors.lastname: Kang
authors.forename: Je Il
authors.initials: JI
authors.lastname: Tak
authors.forename: Woo Young
authors.initials: WY
authors.lastname: Bang
authors.forename: Jae Bok
authors.initials: JB
authors.lastname: Heo
authors.forename: Jin Yong
authors.initials: JY
authors.lastname: Jeong
authors.forename: Gyeong Mee
authors.initials: GM
authors.lastname: Yoon
authors.forename: Ho Young
authors.initials: HY
authors.lastname: Kang
authors.forename: Jeong Dong
authors.initials: JD
authors.lastname: Bahk
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism; D016984:Enterococcus faecium / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C048817:RepB protein, Bacteria