In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

Abstract

Using a mixture of 10 purified DNA replication and DNA recombination proteins encoded by the bacteriophage T4 genome, plus two homologous DNA molecules, we have reconstituted the genetic recombination–initiated pathway that initiates DNA replication forks at late times of T4 bacteriophage infection. Inside the cell, this recombination-dependent replication (RDR) is needed to produce the long concatemeric T4 DNA molecules that serve as substrates for packaging the shorter, genome-sized viral DNA into phage heads. The five T4 proteins that catalyze DNA synthesis on the leading strand, plus the proteins required for lagging-strand DNA synthesis, are essential for the reaction, as are a special mediator protein (gp59) and a Rad51/RecA analogue (the T4 UvsX strand-exchange protein). Related forms of RDR are widespread in living organisms—for example, they play critical roles in the homologous recombination events that can restore broken ends of the DNA double helix, restart broken DNA replication forks, and cross over chromatids during meiosis in eukaryotes. Those processes are considerably more complex, and the results presented here should be informative for dissecting their detailed mechanisms.