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Complementation assay of primer protein: gene expression systems of plasmid vectors support the infection of
T Kishi1, K Miura, K Matsumoto
1Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Abstract:
Two different expression systems of genes of primer proteins (pE for phage M2, and p3 for phi 29) were constructed to study the protein primed DNA replication of Bacillus phages M2 and phi 29. In one system, expression of the genes was under the control of the inducible spac promoter, whereas in the other system, the expression was under the control of the constitutive promoter in plasmid pUB110. Complementation tests in vivo were performed between the primer proteins expressed by these systems and mutant phages having suppressor sensitive mutations in the genes of the primer proteins. The phages M2 susE and phi 29 sus3 were complemented by pE and p3 expressed by the systems, respectively. However, the complementation and apparent phage DNA synthesis were not detected in the combinations between susE of phage M2 and p3 of phage phi 29, and vice versa. Although pE and p3 proteins exhibited structurally and functionally similar characteristics, these proteins showed species specificity in the protein primed DNA replication of bacteriophages M2 and phi 29.
Insights
Primer proteins pE and p3 are essential for Bacillus phage DNA replication. These proteins show species specificity, meaning pE functions with phage M2 and p3 with phage phi 29, but not vice versa.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophages utilize protein-primed DNA replication.
- Primer proteins are crucial for initiating this process.
- Phages M2 and phi 29 are models for studying protein-primed replication.
Purpose of the Study:
- To investigate the function and specificity of primer proteins pE (phage M2) and p3 (phage phi 29).
- To construct expression systems for studying these primer proteins.
- To determine if primer proteins exhibit species specificity in DNA replication.
Main Methods:
- Construction of inducible (spac promoter) and constitutive (pUB110 promoter) gene expression systems for primer proteins.
- In vivo complementation tests using mutant phages (M2 susE, phi 29 sus3) and expressed primer proteins.
- Assessment of phage DNA synthesis.
Main Results:
- Expressed pE complemented M2 susE phage, and expressed p3 complemented phi 29 sus3 phage.
- No complementation or apparent DNA synthesis was observed between M2's pE and phi 29's p3, or vice versa.
- pE and p3 proteins share structural and functional similarities.
Conclusions:
- Primer proteins pE and p3 demonstrate species specificity in the protein-primed DNA replication of Bacillus phages M2 and phi 29.
- Despite functional similarities, cross-complementation between heterologous primer proteins and phages does not occur.
- This specificity is a key characteristic of protein-primed DNA replication in these bacteriophages.