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Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide
H Kobayashi1, K Kitabayashi, K Matsumoto
1Life Science Institute, Sophia University, Tokyo, Japan.
Abstract:
Primer protein (PP) of bacteriophages M2 and phi 29 contains an Arg-Gly-Asp (RGD) sequence. The RGD-mediated protein-protein interaction in protein-primed DNA replication of M2 was studied in vitro using three purified and indispensable components: PP, DNA polymerase (POL) and template DNA linked to terminal protein (TP). PP competed with a synthetic RGD peptide for binding to the template DNA-TP complex (TP-DNA). In addition, POL bound to template TP-DNA only when complexed with PP. These results indicate that the RGD sequence of PP is responsible for the interaction of the PP-POL complex with TP-DNA, which contains the initiation site for the protein priming of DNA synthesis. At the moment when PP converts to TP upon linking the first deoxynucleotide, a conformational change results in exposure of the RGD binding site.
Insights
Primer protein (PP) interactions are key to protein-primed DNA replication. The Arg-Gly-Asp (RGD) sequence in PP mediates binding to template DNA-TP, enabling DNA polymerase (POL) activity.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage DNA replication often utilizes a protein primer.
- Primer protein (PP) in bacteriophages M2 and phi 29 possesses an Arg-Gly-Asp (RGD) sequence.
Purpose of the Study:
- To investigate the role of the RGD sequence in PP-mediated protein-protein interactions during M2 bacteriophage DNA replication.
- To elucidate the mechanism by which PP facilitates DNA polymerase (POL) binding to the template DNA-terminal protein (TP) complex.
Main Methods:
- In vitro studies using purified components: PP, POL, and TP-DNA complex.
- Competition assays with synthetic RGD peptides to assess binding interactions.
- Analysis of POL binding to TP-DNA in the presence and absence of PP.
Main Results:
- PP directly competed with synthetic RGD peptides for binding to the TP-DNA complex.
- DNA polymerase (POL) binding to the TP-DNA template was dependent on the presence of PP.
- The RGD sequence of PP was identified as crucial for the interaction between the PP-POL complex and the TP-DNA initiation site.
Conclusions:
- The RGD sequence in bacteriophage PP is essential for initiating protein-primed DNA replication.
- PP acts as a bridge, facilitating POL recruitment to the TP-DNA complex via its RGD motif.
- A conformational change in PP upon initiation of DNA synthesis exposes the RGD binding site.