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 T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior 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 inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient 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 interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.
Birkenkamp-Demtröder
K
K
Institut für Genetik der Universität zu Köln, Germany.
Golz
S
S
Kemper
B
B
D017122:Bacteriophage T4 / Q000201:enzymology*; D002846:Chromatography, Affinity; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000378:metabolism; D006868:Hydrolysis; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII