Abstract
Protein-mediated exchange of homologous DNA strands is a central reaction in general genetic recombination and the mechanism by which proteins mediate this process in vivo is a topic of keen interest. The dda protein of the bacteriophage T4 is a DNA helicase that has been shown to accelerate branch migration catalyzed by the phage uvsX and gene 32 proteins in vitro (Kodadek, T., and Alberts, B.M. (1987) Nature 326, 312-314). This study did not address the potential role of the helicase in protein-mediated homologous pairing, the first phase of the overall strand-exchange reaction. It is shown here that the dda protein inhibits uvsX protein-mediated pairing between homologous single and double-stranded DNAs. Experiments using deproteinized heteroduplex joints demonstrate that the dda helicase is capable of unwinding these structures to some extent and suggests that this activity may be responsible for the observed inhibition of pairing. It is found that the helicase also reduces the level of uvsX protein-mediated, single-stranded DNA-dependent ATP hydrolysis in the strand-exchange reactions, suggesting that the helicase may also act to destabilize the uvsX protein-DNA filaments that are important intermediates in the pairing reaction. Three other helicases are found to have no effect on the uvsX protein-mediated pairing reaction. A model rationalizing the ability of the dda protein to both inhibit homologous pairing and stimulate branch migration is presented and possible in vivo roles for this interesting activity are discussed.
Highlights
ATP hydrolysis in the strand-exchange reactions, sugt-o 3‘ polarity, stimulates uvsX protein-mediated branchmigesting that thehelicase may alsoact to destabilizethe gration [15].Its failure to influence the rate of the analogous uvsX protein-DNA filaments thatare important inter- recA protein-catalyzed process suggests that the direct promediates in the pairing reaction
When purified dda protein is present in the reaction mixture, a lower rate of pairing is observed (Fig. 1)
In the absence of the helicase, all of the linear duplex DNA is incorporated into paired products within 1min, while in the presence of the dda protein, more than half of the duplex is unreacted a t this time and the reaction approaches completion only after about 2 min
Summary
From the Departmentof Chemistry and Biochemistry and the Clayton Foundation Biochemical Institute, Universiotyf Texas, Austin, Texas 78712. The ability of RecA, UvsX, and deproteinized heteroduplex joints demonstrattheat the yeast strand transferases to mediate homologous pairing is dda helicase is capable of unwinding these structures strongly stimulatedby single-stranded DNA-binding proteins to some extent and suggests that this activity may be [1, 3, 9,10,11]. The T4 system has been especially usefulfor responsible for the observed inhibitioonf pairing It is identifying other potential componeonftsthe strandexchange found that the helicase reduces the level of uvsX machine [12,13,14]. ATP hydrolysis in the strand-exchange reactions, sugt-o 3‘ polarity, stimulates uvsX protein-mediated branchmigesting that thehelicase may alsoact to destabilizethe gration [15].Its failure to influence the rate of the analogous uvsX protein-DNA filaments thatare important inter- recA protein-catalyzed process suggests that the direct promediates in the pairing reaction.
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