Mutations affecting lysine-35 of gpNu1, the small subunit of bacteriophage lambda terminase, alter the strength and specificity of holoterminase interactions with DNA

Virology
Y Hwang, M Feiss

Abstract

The small subunit of lambda terminase, gpNu1, contains a low-affinity ATPase activity that is stimulated by nonspecific dsDNA. The location of the gpNu1 ATPase center is suggested by a sequence match between gpNu1 (29-VLRGGGKG-36) and the phosphate-binding loop, or P-loop (GXXXXGKT/S), of known ATPase. The proposed P-loop of gpNu1 is just downstream of a putative helix-turn-helix DNA-binding motif, located between residues 5 and 24. Published work has shown that changing lysine-35 of the proposed P-loop of gpNu1 alters the response of the ATPase activity to DNA, as follows. The changes gpNu1 k35A and gpNu1 K35D increase the level of DNA required for maximal stimulation of the gpNu1 ATPase by factors of 2- and 10-fold, respectively. The maximally stimulated ATPase activities of the mutant enzymes are indistinguishable from that of the wild-type enzyme. In the present work, the effects of changing lysine-35 on the cos-cleavage and DNA-packaging activities of terminase were examined. In vitro, the gpNu1 K35A enzyme cleaved cos as efficiently as the wild-type enzyme, but required a 2-fold increased level of substrate DNA for saturation, suggesting a slight reduction in DNA affinity. In a crude DNA-packaging system using cleaved lam...Continue Reading

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Citations

Feb 22, 2003·Nucleic Acids Research·Brigitte ScholzElke Bogner
Aug 9, 2008·Annual Review of Genetics·Venigalla B Rao, Michael Feiss
Mar 1, 2005·Journal of Molecular Biology·Alok Dhar, Michael Feiss
Jun 7, 2002·Molecular Cell·Tonny de BeerCarlos Enrique Catalano

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