INHIBITION OF HOLLIDAY STRUCTURE RESOLVING ENDONUCLEASE-VII OF BACTERIOPHAGE-T4 BY RECOMBINATION ENZYMES UVSX AND UVSY

Proteins UvsX, UvsY and Endonuclease VII (endo VII) of bacteriophage T 4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of bran ches from newly made DNA, Frier to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates , generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibit ory effect of UvsX or UvsY is also seen in reactions with Endo VII usi ng two synthetic cruciform DNAs and a C/C-mismatch containing substrat e. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were suffic ient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein int eraction between Endo VII, UvsX and UvsY as suggested by electrophoret ic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immob ilized on a NHS-activated Sepharose matrix. This is the first identifi cation of phage-encoded proteins which modulate the potent endonucleol ytic activity of gp49 in vitro. (C) 1997 Academic Press Limited.