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.
Gual
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A
Departamento de Biotecnologia Microbiana, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.
Camacho
A G
AG
Alonso
J C
JC
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