ABSTRACT
Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.
Subject(s)
Bacteriophage T4/genetics , DNA Damage , Genome , Recombination, Genetic , Replication Origin , Amino Acid Sequence , Base Sequence , Electrophoresis, Gel, Two-Dimensional , Models, Genetic , Molecular Sequence Data , Mutation , Plasmids/genetics , Plasmids/metabolism , Point Mutation , Ultraviolet RaysABSTRACT
Two-dimensional gel analysis of the bacteriophage T4 ori(uvsY) region revealed a novel "comet" on the Y arc. This comet contains simple Y molecules in which the branch points map to the ori(uvsY) transcript region. The comet depends on the the origin and DNA synthesis and is abolished by a mutation that reduces replication without affecting transcription. These results argue that the branched molecules are intermediates in replication initiation. A transcriptional terminator, cloned just downstream of the origin promoter, shortened the tail of the comet. Therefore, the location of the transcript determines the DNA branch points. We conclude that the comet DNA consists of intermediates in which unidirectional replication has been triggered by priming from the RNA of the origin R loop.
Subject(s)
Bacteriophage T4/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase , Replication Origin/physiology , Virus Replication/physiology , Bacteriophage T4/genetics , Blotting, Northern , Blotting, Southern , DNA Helicases/metabolism , DNA Replication/genetics , DNA, Viral/biosynthesis , DNA, Viral/isolation & purification , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Electrophoresis, Gel, Two-Dimensional , Escherichia coli/virology , Membrane Proteins/genetics , Membrane Proteins/physiology , Mutation , Physical Chromosome Mapping , RNA, Viral/metabolism , Recombination, Genetic/physiology , Replication Origin/genetics , Ribonuclease H/genetics , Ribonuclease H/physiology , Terminator Regions, Genetic/genetics , Transcription, Genetic/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/physiology , Virus Replication/geneticsABSTRACT
The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.
