The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1.
Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 A crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 A out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.
Nash
Rebekah Potts
RP
Department of Chemistry, University of North Carolina, Chapel Hill, CB 3290 and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, CB 7260, Chapel Hill, NC 27599, USA.
Habibi
Sohrab
S
Cheng
Yuan
Y
Lujan
Scott A
SA
Redinbo
Matthew R
MR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003227:Conjugation, Genetic; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; D008670:Metals; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase
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