Mutations affecting lysine-35 of gpNu1, the small subunit of bacteriophage lambda terminase, alter the strength and specificity of holoterminase interactions with DNA.
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 lambda DNA as substrate, the gpNu1 K35A enzyme had a 10-fold defect. In vivo, lambda Nu1 K35A showed a 2-fold reduction in cos cleavage, but no packaged DNA was detected. The primary defect of the gpNu1 K35A enzyme was concluded to be in a post-cos-cleavage step of DNA packaging. In in vitro cos-cleavage experiments, the gpNu1 K35D enzyme had a 10-fold increased requirement for saturation by substrate DNA. Furthermore, the cos-cleavage activity of gpNu1 K35D enzyme was strongly inhibited by the presence of nonspecific DNA, indicating that the gpNu1 K35D enzyme is unable to discriminate effectively between cos and nonspecific DNA. No cos cleavage was observed in vivo for lambda Nu1 K35D, a result consistent with the discrimination defect found in vitro for the gpNu1 K35D enzyme. In a crude packaging system the gpNu1 K35D enzyme had a 200-fold defect; in a purified packaging system, the gpNu1 K35D enzyme was found to be unable to discriminate between lambda DNA and nonspecific phage T7 DNA, a result indicating that the gpNu1 K35D enzyme is also defective in discriminating between lambda DNA and nonspecific DNA during DNA packaging.
Hwang
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Y
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Feiss
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M
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005814:Genes, Viral; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004706:Endodeoxyribonucleases; C030894:terminase