Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

• Published in: The Journal of biological chemistry 2000-11, Vol.275 (45), p.35311-35319
• Main Authors: Gual, Aranzazu, Camacho, Ana G., Alonso, Juan C.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - physiology; Adenosine Triphosphate - metabolism; Bacillus subtilis; Bacillus subtilis - chemistry; Bacteriophages - chemistry; Bacteriophages - physiology; Base Sequence; distamycin A; Distamycins - pharmacology; DNA - metabolism; DNA, Superhelical - metabolism; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - physiology; Endonucleases - metabolism; Escherichia coli - metabolism; Hydrolysis; Kinetics; Models, Biological; Models, Genetic; Molecular Sequence Data; Phage SPP1; Plasmids - metabolism; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; terminase; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004309200

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5′-RCGG↓CW-3′ sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at bothbona fide 5′-CTATTGCGG↓C-3′ sequences withinpacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and Pi. H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PΔN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.