Single-molecule analysis of protein·DNA complexes formed during partition of newly replicated plasmid molecules in Streptococcus pyogenes

Florencia Pratto, Yuki Suzuki, Kunio Takeyasu, Juan C. Alonso

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

The Streptococcus pyogenes pSM19035 partition locus is ubiquitous among plasmids from vancomycin- or methicillin-resistant bacteria. An increasing understanding of this segregation system may highlight novel protein targets that could be blocked to curb bacterial proliferation. pSM19035 segregation depends on two homodimeric (δ2 (ParA) and ω2 (ParB)) proteins and six cis-acting centromeric noncurved parS sites. In the presence of ATP·Mg2+, δ2 (δ·ATP·Mg2+)2 binds DNA in a sequence-independent manner. Protein ω2 binds with high affinity and cooperatively to B-form parS DNA. Atomic force microscopy experiments indicate that about 10 ω2 molecules bind parS, consisting of 10 contiguous iterons. Protein (δ·ATP·Mg2+)2, by interacting with the N terminus of ω2 bound to parS, loses its association with DNA and relocalizes with ω 2·parS to form a ternary complex ((δ·ATP·Mg2+)2·ω 2·parS) with the DNA remaining in straight B-form. Then, the interaction of two (δ·ATP·Mg2+)2·ω 2·parS complexes via δ2 promotes pairing of a plasmid subfraction. (δD60A·ATP·Mg2+)2, which binds but does not hydrolyze ATP, leads to accumulation of pairing intermediates, suggesting that ATP hydrolysis induces plasmid separation. We propose that the molar ω22 ratio regulates the different stages of pSM19035 segregation, pairing, and δ2 polymerization, before cell division.

Original languageEnglish
Pages (from-to)30298-30306
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number44
DOIs
Publication statusPublished - 2009 Oct 30

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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