In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Poxvirus infection involves the conversion of viral genomes and transfected DNA into high-molecular-weight concatemers.
• The precise mechanism and protein catalysts responsible for viral DNA concatemer formation remain largely unknown, despite its association with viral replication.

Purpose of the Study:

• To elucidate the mechanism and identify the protein(s) responsible for viral DNA concatemer formation.
• To investigate the role of vaccinia virus DNA polymerase in catalyzing DNA joining reactions.
• To explore the potential of this reaction for DNA cloning applications.

Main Methods:

• In vitro assays using vaccinia virus DNA polymerase to assess its ability to catalyze single-stranded annealing reactions.
• Characterization of concatemer formation using linear duplex DNA substrates with varying end structures and homology.
• Investigation of the role of vaccinia single-stranded DNA-binding protein (gpI3L) and nucleolytic processing by the 3'-to-5' proofreading exonuclease.

Main Results:

• Vaccinia virus DNA polymerase was shown to catalyze single-stranded annealing, forming linear or circular concatemers from linear duplex substrates.
• The reaction requires at least 12 base pairs of shared sequence at DNA ends and is stimulated by vaccinia single-stranded DNA-binding protein (gpI3L).
• Duplex joining is dependent on nucleolytic processing by the 3'-to-5' proofreading exonuclease and is inhibited by dNTPs; concatemers are initially noncovalently joined.

Conclusions:

• The study identifies vaccinia virus DNA polymerase as a key enzyme in viral DNA concatemer formation via single-stranded annealing.
• The findings provide a mechanistic basis for understanding viral replication and offer a novel method for cloning PCR-amplified DNA.
• Noncovalently joined concatemers can be stabilized in E. coli if homologies permit circular molecule formation.