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J. Biol. Chem., Vol. 277, Issue 45, 42645-42653, November 8, 2002

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Structure-Function Analysis of Escherichia coli DNA Helicase I Reveals Non-overlapping Transesterase and Helicase Domains^*

Devon R. Byrd^§, Juliana K. Sampson, Heather M. Ragonese, and Steven W. Matson^¶**

From the  Department of Biology, the ^¶ Curriculum in Genetics and Molecular Biology, and the  Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-3280

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of ~650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.

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