THE TRAY GENE-PRODUCT AND INTEGRATION HOST FACTOR STIMULATE ESCHERICHIA-COLI DNA HELICASE I-CATALYZED NICKING AT THE F-PLASMID ORIT

F plasmid conjugative transfer is initiated by the introduction of a s ite- and strand-specific nick within the plasmid origin of transfer (o riT), Genetic studies have shown nick formation to be dependent on bot h the traI and traY genes, However, highly purified TraIp, the traI ge ne product, nicks oriT in a site- and strand-specific manner in the ab sence of the traY gene product (TraYp) in vitro (Matson, S. W., and Mo rton, B. S. (1991) J. Biol, Chem. 266, 16232-16237). Analysis of the o riT region has revealed binding sites for TraYp and the host protein i ntegration host factor (IHF). To explore possible interactions occurri ng at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate, A marked enhancement of t he nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF, In addition, TraIp was able to nick a li near oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone, Individual protein concentration requirements for the supercoil ed and linear nicking reactions were similar, and the reactions occurr ed at equal velocity, suggesting that they are biochemically identical , Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported K-D values for the I HF and TraYp binding sites in oriT, These data, coupled with data pres ented in the accompanying report, suggest that TraYp and IHF bind inde pendent of one another, forming a nucleo-protein complex with oriT tha t can be recognized and nicked by TraIp.