The determination of functional amino acid residues for F TraI relaxase activity and specificity

TraI is an essential protein for bacterial conjugation, a process whereby a plasmid directs its transfer from a donor to a recipient bacterium. The TraI proteins from the plasmids F and R100 have two functions, a single-stranded DNA endonuclease (or “relaxase”) activity and a helicase activity. Based on limited proteolysis experiments of F TraI, constructs that encode the relaxase domains from the two TraI homologues (TraI36) were generated. Two mutagenesis-based strategies were undertaken to identify functionally important amino acid residues of the F TraI relaxase domain. The first strategy was alanine scanning mutagenesis to determine the importance of thirteen amino acids conserved among F-like relaxases. Several amino acids have been identified that are involved in binding to the plasmid origin of transfer, and two histidines have been identified as participants in the ssDNA cleavage reaction. The second strategy was aimed at identifying the residues of F and R100 TraI36, which are 91% identical in primary sequence, that are responsible for the two-base difference in the DNA binding specificity of these two homologues. Amino acids 193 and 201 were identified as the major specificity determinants of the differential ssDNA binding and cleavage activities of F and R100 TraI36. The studies presented in this dissertation increase our understanding of protein-ssDNA binding interactions and specificities, and form the beginning of an active site map for the F TraI relaxase.