Analysis of processivity of mungbean dideoxynucleotide-sensitive DNA polymerase and detection of the activity and expression of the enzyme in the meristematic and meiotic tissues and following DNA damaging agent.
Analysis of the processivity of mungbean ddNTP-sensitive DNA polymerase showed the incorporation of approximately 35-40 nucleotides per binding event in the replication assays involving M13 ss DNA template with 5'-labeled 17-mer primer. Optimal processivity was obtained with 100-150 mM KCl and 6-8 mM Mg2+ at pH 7.5. The enzyme showed preference for Mg2+ over Mn2+ as the metal activator for processivity. 2', 3' dideoxythymidine 5' triphosphate (ddTTP) and rat DNA pol beta antibody strongly influenced distributive synthesis. Considerable enhancement in processivity was noticed at 1mM ATP and 2-4 mM spermidine while higher concentrations of spermidine caused distributive synthesis. The enzyme was found to be active in both meristematic and meiotic tissues and distinctly induced by EMS treatment. DNA-binding assays revealed distinct binding ability of the enzyme to template/primer and damaged DNA substrate. Together these observations illustrate the probable involvement of the enzyme in replication and repair machinery in higher plants.
Roy
Sujit
S
Protein Chemistry laboratory, Department of Chemistry, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Kolkata 700 009, West Bengal, India.
Choudhury
Swarup Roy
SR
Sengupta
Dibyendu N
DN
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008540:Meiosis; D018519:Meristem / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013696:Temperature
D008345:Manganese; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D005020:Ethyl Methanesulfonate; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D013095:Spermidine