Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1.
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.
Kim
Sam Woong
SW
Division of Applied Life Sciences, Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea.
Jeong
Eun Ju
EJ
Kang
Han Soo
HS
Tak
Je Il
JI
Bang
Woo Young
WY
Heo
Jae Bok
JB
Jeong
Jin Yong
JY
Yoon
Gyeong Mee
GM
Kang
Ho Young
HY
Bahk
Jeong Dong
JD
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
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C048817:RepB protein, Bacteria