Functional analysis of the terminase large subunit, G2P, of Bacillus subtilis bacteriophage SPP1.
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 downward arrowCW-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 both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (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.
Departamento de Biotecnologia Microbiana, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.
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D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry*; D001435:Bacteriophages / Q000737:chemistry* / Q000502:physiology*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004214:Distamycins / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000502:physiology; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004214:Distamycins; C417248:bacteriophage SPP1 terminase; C030000:stallimycin; D000255:Adenosine Triphosphate; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C030894:terminase; D000251:Adenosine Triphosphatases