Replication of Bacteriophage ϕX174 Replicative Form DNA In Vivo

Summary

The replication of the circular double-stranded bacteriophage φX174 replicative form DNA was studied by structural analysis of pulse-labeled replicative intermediates. Evidence is presented that φX replicative form replicates according to a rolling circle model proposed by Dressier & Wolfson (1970). Replication involves continuous elongation of the viral (= positive) strand component of replicative form resulting in the displacement of a single-stranded tail of increasing length. Replicative intermediates sedimenting at 27 to 28 s are found to contain linear viral strands of approximately double φX unit length. The synthesis of the new complementary (= negative) strand on the single-stranded tail appears to be initiated with considerable delay and converts the tail to double-stranded DNA. Before the new negative strand is completed, the replicative intermediates split into (I) a complete RF molecule containing the “old” negative and the “new” positive strand and (il) a linear partially double-stranded “tail” consisting of the complete “old” positive strand and a fragment of the “new” negative strand.

The second part of this study is concerned with the fate during RF replication of these fragments of the rolling circles. The RF II molecules containing the “old” negative strands appear to go into further replication rounds repeatedly. Some of the “tails” were found in the infected cells in their original linear form. “Gapped” RF II molecules which have been described earlier by Schekman and coworkers (1971) are supposed to originate from the tails of rolling circle intermediates by circularization of their positive strand components. Evidence is provided by our experiments that even late during RF replication the gaps exist in the negative strands of RF II rather exclusively. Appropriate chase experiments indicated that the “tails” finally are converted to RF I molecules. Progeny RF I molecules could not be observed to start new replication rounds under our conditions although we cannot exclude that this might happen to some minor extent.

The results presented suggest that the first negative strands rather than the parental positive strands persist as master templates during φX RF replication.