Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex.
The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.
D010584:Bacteriophage phi X 174 / Q000235:genetics* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D003852:Deoxyribonucleoproteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004279:DNA, Viral; D003852:Deoxyribonucleoproteins