Electrophoretic characterization of intracellular forms of bacteriophage phi X174 DNA: identification of novel intermediate of altered superhelix density

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Abstract
The replication cycle of bacteriophage .vphi.X174 DNA was analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and 40 min after infection was deduced and quantitated. Migration through 1.4% agarose at 5 and 10 V/cm resolved all known viral DNA species as well as fragments of host [Escherichia coli] chromosomal DNA. Among parental replicative form (RF) molecules synthesized, 1-3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most input viral strands remained in a duplex structure throughout the infection period. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF existed as form I DNA. Approximately 1% of the viral DNA was found as unit length linear single strands. Electrophoretic analysis of RF DNA after controlled denaturation suggests the existence of 4 populations of closed circular RF: molecules of native superhelix density (RFI); a population of molecules of altered topological linking number, .alpha., differing in increments of 1 superhelical turn (.tau.) between .tau. values of 0 and approximately -31; a superimposed population of topological isomers which under electrophoresis conditions have a mean .tau. value (.hivin..tau.) equal to +5; and a population of complexed molecules with a reduced number of superhelical turns due to their association with single-stranded DNA and RNA. Complexed parental molecules isolated from cells infected at high multiplicity release RFI and homologous single-stranded DNA upon denaturation and may be intermediates in genetic recombination. Complexed RF DNA isolated from cells infected at low multiplicity release native supercoils upon reaction with RNase H and are observed by EM to contain displacement loops. Such molecules are likely intermediates in transcription. The results are consistent with a structure of complexed RFI involving a partially triple-stranded helix in which a covalently closed circular duplex molecule contains a reduced number of superhelical turns due to the unwinding produced by base pairing between 1 strand of the supercoil and an associated homologous single strand of DNA or RNA.