Abstract
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 iteron-carrying theta-type plasmids.
| Original language | English |
|---|---|
| Pages (from-to) | 1841-1847 |
| Number of pages | 7 |
| Journal | Journal of Microbiology and Biotechnology |
| Volume | 17 |
| Issue number | 11 |
| Publication status | Published - 2007 Nov 1 |
Fingerprint
Keywords
- Enterococcus faecalis
- RepA
- Replication
- Replication initiator
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
- Microbiology
- Bioengineering
Cite this
Molecular interactions of a replication initiator protein, RepA, with the replication origin of the enterococcal plasmid p703/5. / Cha, Kyung Il; Lim, Kihong; Jang, Sehwan; Lim, Wangjin; Kim, Taehyung; Chang, Hyo-Ihl.
In: Journal of Microbiology and Biotechnology, Vol. 17, No. 11, 01.11.2007, p. 1841-1847.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Molecular interactions of a replication initiator protein, RepA, with the replication origin of the enterococcal plasmid p703/5
AU - Cha, Kyung Il
AU - Lim, Kihong
AU - Jang, Sehwan
AU - Lim, Wangjin
AU - Kim, Taehyung
AU - Chang, Hyo-Ihl
PY - 2007/11/1
Y1 - 2007/11/1
N2 - 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 iteron-carrying theta-type plasmids.
AB - 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 iteron-carrying theta-type plasmids.
KW - Enterococcus faecalis
KW - RepA
KW - Replication
KW - Replication initiator
UR - http://www.scopus.com/inward/record.url?scp=36949022484&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36949022484&partnerID=8YFLogxK
M3 - Article
VL - 17
SP - 1841
EP - 1847
JO - Journal of Microbiology and Biotechnology
JF - Journal of Microbiology and Biotechnology
SN - 1017-7825
IS - 11
ER -