Single-stranded DNA binding by F TraI relaxase and helicase domains is coordinately regulated

J Bacteriol. 2010 Jul;192(14):3620-8. doi: 10.1128/JB.00154-10. Epub 2010 Apr 30.

Abstract

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal approximately 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Nucleotidyltransferases / metabolism*
  • DNA, Single-Stranded / physiology*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Gene Expression Regulation, Bacterial / physiology
  • Protein Binding
  • Protein Structure, Tertiary

Substances

  • DNA, Single-Stranded
  • Escherichia coli Proteins
  • DNA Nucleotidyltransferases
  • DNA relaxase
  • TraI protein, E coli
  • DNA Helicases