Abstract
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.
Highlights
T o be necessary for DNA The traM gene product has thenic site withinoriT, but tion ofnovelrecombinantproductsresultingfrom a isnotrequired for nicking to occur [7, 8]
tra plasmid-encodedtrul gene product (TraIp)-catalyzedcleavagefligationreactionF.urtherbiochemical role has been assigned to the traD gene product, more, the cleavage and ligation reactions were boseth- it has been identified as an inner membraneprotein quence-specific
Molecular genetic studies of the Escherichia coli F plasmid have described24geneproducts of the tra family that are involved in conjugative DNA transfer from a donor F’ cell to a recipient F- cell [1,4]
Summary
The sequences for the oligonucleotidesused in this study are shown. Each oligonucleotidewas named based on its length in nucleotides. Vised using single-stranded DNA (ssDNA)' oligonucleotides containing the oriT nic site. This assay was based on methods employed t o investigate the possibility of a cleavagehigation activity for the TraIp-like proteins of other transmissableplasmids [18,19]. When 5'-end labeled oligonucleotides wereused, the reactions were terminated as described above forthe cleavage reactions. One set of reactions was heated at 65 "C for 20 min prior to otides were either labeled on the 5'-end using bacteriophage T4 loading on the gel, and one set of reactions was directly loaded onthe polynucleotidekinase and [y32PlATPas described [20]or on the 3'-end using terminal deoxynucleotidyl transferase and [ Otides by gel filtration chromatography using a 1.5-ml Sephadex G-50 column (0.6cm x 7.5 cm). [y-32PlATaPnd [ C Y - ~ ~ P I ~w~eAreTfrPomAm-
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