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Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29
G Martín1, J M Lázaro, E Méndez
1Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Abstract:
The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.
Insights
Phage phi 29 protein p5 enhances phi 29 DNA replication by binding single-stranded DNA. This protein stimulates replication when it would normally plateau, indicating a crucial role in DNA synthesis.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Phage phi 29 is a model organism for studying DNA replication.
- Protein p5 is essential for the elongation phase of phi 29 DNA replication in vivo.
Purpose of the Study:
- To highly purify and characterize phage phi 29 protein p5.
- To investigate the function of protein p5 in phi 29 DNA replication.
Main Methods:
- High-level purification of protein p5 from E. coli and B. subtilis.
- Amino acid analysis and N-terminal sequencing for protein identification.
- DNA binding assays and nuclease protection experiments.
Main Results:
- Purified protein p5 binds to single-stranded DNA and protects it from degradation.
- Protein p5 does not affect initiation complex formation or elongation rate.
- Protein p5 significantly stimulates phi 29 DNA-protein p3 replication at later incubation times.
Conclusions:
- Phage phi 29 protein p5 plays a critical role in enhancing DNA replication efficiency.
- Protein p5's ability to bind and protect single-stranded DNA likely contributes to its stimulatory effect on replication.