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

Abstract

The replication cycle of bacteriophage phi X174 DNA has been analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and40 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 chromosomal DNA. Among parental replicative form(RF) molecules synthesized, 1 to 3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most of the input viral strands remained in a duplex structure throughout the period of infection studied here. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF were found to exist as form I dna. in addition, 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 four populations of closed circular RF: (i) molecules of native superhelix density (RFI); (ii) a population of molecules of altered topological linking number, alpha, differing in increments of one superhelical turn (tau) between tau values of 0 and approximately -31; (iii) a superimposed population of topological isomers which under electrophoresis conditions have mean tau value (tau) equal to +5; and (iv) 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 released FRI and homologous single-stranded DNA upon denaturation and are postulated to 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 electron microscopy to contain displacement loops. Such molecules are likely intermediates in transcription. Our 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 one strand of the supercoil and an associated homologous single strand of DNA or RNA.