The bacteriophage phi29 packaging proteins supercoil the DNA ends.
Bacteriophage phi29 DNA with covalently bound terminal protein (DNA-gp3) and its left and right-end restriction fragments (L and R-DNA-gp3) sedimented faster in sucrose density gradients than their proteinase K-treated counterparts, and the faster sedimentation was both gp3 and Mg2+-dependent. Addition of gp16, the phi29 DNA packaging ATPase, further increased the sedimentation rates of both intact DNA-gp3 and L and R-DNA-gp3 fragments. Thus, DNAs with gp3 were more compact than gp3-free DNA, and gp16 further condensed the DNA-gp3 forms. [35S]gp16 cosedimented with the fast-sedimenting DNA-gp3 fragments, and the putative L-DNA-gp3-gp16 complexes were packaged preferentially into proheads in the defined in vitro system. Lariats of DNA-gp3 and L and R-DNA-gp3 observed by electron microscopy rationalized the sedimentation results, and lariats with multiple loops or coils increased tenfold in a preparation of L-DNA-gp3-gp16 complexes. The rapid sedimentation and the structure of the DNA-gp3-gp16 complexes were consistent with supercoiling of lariat loops, and treatment with topoisomerase I shifted fast-sedimenting complexes toward the uncoiled lariat position in sucrose density gradients. DNA-gp3 has a maturation pathway in which the packaging proteins gp3 and gp16 supercoil the DNA ends, probably as a prerequisite for efficient interaction with the prohead.
Grimes
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Department of Microbiology, University of Minnesota, Minneapolis 55455, USA.
Anderson
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D
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017103:Bacillus Phages / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism* / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D004278:DNA, Superhelical; D004279:DNA, Viral; D014764:Viral Proteins; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I