Molecular interactions of a replication initiator protein, RepA, with the replication origin of the enterococcal plasmid p703/5

Kyung Il Cha, Kihong Lim, Sehwan Jang, Wangjin Lim, Taehyung Kim, Hyoihl Chang

Research output: Contribution to journalArticle

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 languageEnglish
Pages (from-to)1841-1847
Number of pages7
JournalJournal of microbiology and biotechnology
Volume17
Issue number11
Publication statusPublished - 2007 Nov

Keywords

  • Enterococcus faecalis
  • RepA
  • Replication
  • Replication initiator

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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