Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.
Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.
Sherman
J A
JA
Department of Biology, University of North Carolina, Chapel Hill 27599.
Matson
S W
SW
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids
D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D004247:DNA; C027093:TraI protein, E coli; D004265:DNA Helicases