Abstract
Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.
Highlights
Protein p5 is a Bacillus subtilis phage (629-encoded protein requiredfor 429 DNA replication in vivoP. rotein p5has single-stranded DNA binding (SSB) capacity and stimulates in vitroDNA replication severalfold when 429 DNA polymerase is used to replicate either the natural
When both 629 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, 429 DNA replication is strongly inhibited. This inhibition is fully overcomeby adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably 429 DNA polymerase, which has a strong affinity for ssDNA
Protein p5 alsboinds to ssDNA in the 429 replicative intermediates produced in vitro, which are similar in structure to those observed in vivoO. ur results strongly suggest that
Summary
The gene coding for one of those proteins, p5, has been cloned, and protein p5 hasbeen overproduced (Martin and Salas, 1988), purified, and shown to have affinity for ssDNA and to stimulate429 DNA replication i n vitro (Martin et al, 1989).it was postulated that p5 might participate aas SSB protein during 429 DNA replication. Proteins that bind stsoDNA with high affinity but without sequence specificity have been purified from several sources (Chase andWilliams, 1986).In addition to binding stsoDNA, tobe considered as SSB replication proteins,theyshould participate in DNA replication They may stimulate their cognateDNA polymerases in stoichiometric quantities with respect to the template, or have an essential role at some stage during genome replication, as is the case for phage fd protein 5 , which controls the switchfrom RF to replication of viral ssDNA (Kornberg, 1980).
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