The Structure of the Plasmid pCU1 TraI Relaxase and the Role of the pCU1 TraI Relaxase-Helicase during Conjugative Plasmid Transfer Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 22, 2019
Creator
  • Nash, Rebekah Potts
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Bacteria disseminate genetic material to neighboring cells using conjugative plasmid transfer (CPT). During CPT, a donor bacterium transfers one strand of a double-stranded DNA plasmid to a recipient. Each conjugative plasmid encodes a complex of proteins necessary for its transfer. One of these proteins, the relaxase, initiates plasmid transfer by severing the nic site of the transferred strand (T-strand). A DNA helicase then separates the T-strand from the parent strand, starting at the nic site. The relaxase acts a second time to terminate transfer by resealing the nicked T-strand. The resistance plasmid pCU1 encodes a multi-domain protein, TraI, that supplies both the relaxase and helicase activities required for its transfer. We analyzed the structure and function of pCU1 TraI in order to compare it to similar plasmid-encoded proteins and to identify TraI-mediated activities that could be targeted by inhibitors. Characterization of the pCU1 relaxase revealed unique structural and functional modifications that this enzyme has introduced into the traditional relaxase-mediated DNA nicking mechanism. First, while the overall fold of the pCU1 relaxase is similar to that of homologous relaxases, its conserved DNA nicking residues (Y18,19,26,27) are flipped up to 14 � out of the relaxase active site. Second, the pCU1 relaxase preferentially utilizes Y26 or a combination of Y18+Y19 when nicking DNA. In contrast, homologous relaxases use the first tyrosine in amino acid sequence for DNA nicking. Third, the pCU1 relaxase lacks the sequence-specific DNA binding characteristic of homologous relaxase enzymes. However, it maintains highly sequence specific and metal-dependent DNA nicking. Analysis of the pCU1 helicase established the extent of the minimal helicase domain, the location of the seven conserved helicase motifs, and the substrate requirements of the helicase ATPase activity. The pCU1 helicase harnesses the energy released during ATP hydrolysis to drive DNA strand separation. After optimizing an ATPase assay for use with pCU1 TraI, small molecule libraries were screened for their ability to inhibit pCU1 TraI, and several potential TraI ATPase inhibitors were identified. In summary, this structural and functional characterization of pCU1 TraI identified features unique to this enzyme and revealed particular activities could be targeted by TraI-specific inhibitors.
Date of publication
Keyword
DOI
Resource type
Rights statement
  • In Copyright
Advisor
  • Redinbo, Matthew R.
Degree
  • Doctor of Philosophy
Graduation year
  • 2011
Language
Publisher
Parents:

This work has no parents.

Items