Mechanism of replication of bacteriophage phi X174 XX. Sensitivity of nascent DNA to single-strand-specific nucleases

Abstract

We reported earlier that dephosphorylated nascent phi X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules. Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size. The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides. Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for [3H]thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease. When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules. We suggest that the resistance of the longer nascent (pronase-treated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end.

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome

“…The molecules were 5' end labeled with 32P by using [.y-32P]ATP and polynucleotide kinase. After separation from the excess [-y-32P]ATP, the DNA was centrifuged on a neutral sucrose gradient, and the sedimentation velocity profile de- (15). TI end labeled with [_y-32P]ATP using polyn nase, precipitated with isopropanol, an graphed on nitrocellulose to remove th( 32P]ATP (18).…”