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Vaccinia virus encodes a polypeptide with DNA ligase activity
Abstract:
Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.
Insights
Vaccinia virus gene SalF 15R encodes a novel DNA ligase. This enzyme is crucial for viral replication and shows increased activity during infection.
Area of Science:
- Molecular Biology
- Virology
- Enzymology
Background:
- Vaccinia virus, a large DNA virus, possesses genes with unknown functions.
- DNA ligases are essential enzymes involved in DNA replication, repair, and recombination.
- The vaccinia virus SalF 15R gene was hypothesized to encode a DNA ligase.
Purpose of the Study:
- To investigate the function of the vaccinia virus SalF 15R gene.
- To identify and characterize a potential DNA ligase encoded by vaccinia virus.
Main Methods:
- Radio-labeling assays using alpha-(32P)ATP to detect DNA ligase activity.
- Enzyme assays to measure DNA ligase activity in infected cell extracts.
- Immunoprecipitation using antiserum against the SalF 15R protein.
Main Results:
- A novel 61 kD radio-labeled polypeptide was detected in vaccinia virus-infected cells incubated with alpha-(32P)ATP.
- This polypeptide formed a covalent adduct with AMP, characteristic of DNA ligases.
- Enzyme activity assays revealed increased DNA ligase activity post-infection.
- Antiserum against SalF 15R immunoprecipitated the 61 kD polypeptide and the DNA ligase-AMP adduct.
Conclusions:
- The vaccinia virus gene SalF 15R encodes a functional DNA ligase.
- This viral DNA ligase is present throughout infection and its activity increases during the infection process.
- The identified DNA ligase is likely involved in viral DNA replication or other essential viral processes.