DNA replication in SV40-infected cells: IX. The inhibition of a gap-filling step during discontinuous synthesis of SV40 DNA

Abstract

SV40 DNA apparently replicates by a discontinuous mechanism similar to that proposed for bacteria and bacteriophages. Transient, 4–5 S oligonucleotide intermediates are formed during a brief pulse-labeling with 3 H-thymidine, and these fragments are rapidly joined to preexisting progeny strands ( Fareed and Salzman, 1972). An inhibitor of DNA synthesis, hydroxyurea, interferes with the joining of these short 4–5 S oligonucleotide intermediates into longer DNA molecules. This results in the accumulation of replicating forms of SV40 DNA with 4–5 S oligonucleotide fragments hydrogen-bonded at the replication forks to parental DNA strands. Replicating molecules of SV40 DNA isolated from lytically infected cells treated with hydroxyurea were repaired in an in vitro DNA synthesizing system using T4 DNA polymerase and Escherichia coli polynucleotide ligase. The experimental results indicate that at least 80% of the short 4–5 S fragments are separated from themselves or longer progeny molecules by single-stranded gaps in the DNA double helix. These gaps can be repaired in vitro, and the fragments ligased to form longer polynucleotide chains. A model for SV40 DNA replication involving two different DNA polymerases is discussed.