44] DNA replication of bacteriophage T4 in Vivo

Abstract

This chapter discusses various methods used to investigate bacteriophage T4 DNA replication in vivo. The T4 genome is large for a bacterial virus. It comprises about 168,800 base pairs. It encodes most of the T4 DNA replication and recombination proteins (except for host RNA polymerase) and enzymes that degrade the host DNA. Two properties of the T4 chromosomes are important when considering DNA replication. First, the ends of individual chromosomes are randomly permuted over the circular map. Second, the glucosylation and hydroxymethylation of the cytosine residues protect T4 DNA from the T4 enzymes that degrade the host DNA, as well as from most of the known restriction enzymes. Origin initiation in most wild-type T4 chromosomes is bidirectional. In primase- or topoisomerase-deficient mutants, initiation from oriA or oriF is unidirectional in the direction of transcription, as expected if transcripts prime leading strand synthesis in one direction, but primase is required to prime Okazaki pieces so that the first Okazaki piece becomes the leading strand in the direction opposite to that of transcription. Probably, topoisomerase has to resolve the ensuing entanglement of DNA strands. The T4 replication origins are mainly characterized by hybridizations of in vivo labeled nascent T4 DNA, isolated early after infection, to Southern blots of cloned T4 fragments or restriction digests of unmodified T4 DNA and by electron microscopy.