Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1

• Published in: Plasmid 2006-03, Vol.55 (2), p.99-113
• Main Authors: Kim, Sam Woong, Jeong, Eun Ju, Kang, Han Soo, Tak, Je Il, Bang, Woo Young, Heo, Jae Bok, Jeong, Jin Yong, Yoon, Gyeong Mee, Kang, Ho Young, Bahk, Jeong Dong
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Base Sequence; DNA Replication - physiology; DNA, Bacterial - metabolism; Enterococcus faecium; Enterococcus faecium - chemistry; Enterococcus faecium - genetics; Enterococcus faecium - isolation & purification; Molecular Sequence Data; Nucleic Acid Conformation; pJB01; Plasmids - biosynthesis; Plasmids - chemistry; Plasmids - isolation & purification; pMV158 family; Protein Binding; RCR; RepB; Repetitive Sequences, Nucleic Acid; Sequence Alignment; Three non-tandem direct repeats
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2005.08.002

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.