New in vitro dna polymerase activity and fidelity assay reveals age-dependent changes in Arabidopsis Thaliana

Abstract

DNA polymerase is an enzyme that adds nucleotides to the growing DNA chain during replication and DNA repair. DNA polymerase activity and fidelity are important characteristics that reflect the ability of DNA polymerase to add nucleotides and then proofread newly synthesized DNA. We have developed a protocol allowing analysis of polymerase activity and fidelity using crude Arabidopsis thaliana plant extracts. It is based on the ability of DNA polymerases in the extract to elongate the fluorescently labelled primer annealed to a short complementary template. For analysis, fluorescently labelled products were separated on a denaturing polyacrylamide gel and visualized using a high performance blot imager. Analysis of tissue prepared from 5-, 12- and 21-day-old Arabidopsis plants showed an age-dependent decrease in polymerase activity, an increase in polymerase fidelity and a tendency to an increase in exo- (endo) nucleolytic activity.