Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage λ and Escherichia coli

Abstract

Initiation of bacteriophage λ DNA replication at the chromosomal origin depends on the λ O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the 'λ single-strand replication reaction', has now been established with eight purified proteins, each of which is also required for replication of the phage λ chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the λ O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of ΦX174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the λ single-strand replication reaction.