Bacteriophage φX174 RF DNA replication in vivo : A study by electron microscopy

We have investigated bacteriophage φX174 RF † DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point. Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication. Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained. The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA. Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo. A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9)