Measurement of kinetic parameters of human platelet DNA polymerase gamma.
Synthesis of mitochondrial DNA is performed by DNA polymerase gamma. Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, are a major cause of neurological disease. A large proportion of patients carry rare nucleotide substitutions leading to single amino acid changes. Confirming that these replacements are pathogenic can be problematic without biochemical evidence. Here, we provide a hands-on protocol for an in vitro kinetic assay of DNA polymerase gamma which allows assessment of the K(m) and V(max) for the incoming nucleotide of the polymerization reaction. To avoid measurement of contaminating nuclear DNA polymerases, platelet extracts are used since platelets do not contain a nucleus. Moreover, platelets have the advantage of being obtainable relatively non-invasively. Polymerization activity is determined by measurement of the incorporation of radioactive thymidine 5'-triphosphate (dTTP) on the homopolymeric RNA substrate poly(rA).oligo(dT)(12-18). To further minimize nuclear DNA polymerase activity, aphidicolin, an inhibitor of most nuclear DNA polymerases, is included in the reaction. In addition, reactions are carried out in the absence and presence of the competitive inhibitor of DNA polymerase gamma, 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), to allow calculation of the ddTTP-sensitive incorporation. With this method, platelets from healthy control subjects extracted with 3% Triton X-100 showed a K(m) for dTTP of 1.42 microM and a V(max) of 0.83 pmol min(-1)mg(-1).
Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, United Kingdom. j.taanman@medsch.ucl.ac.uk
Jan-Willem
JW
Taanman
Margit
M
Heiske
Thierry
T
Letellier
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000097:blood*; D054306:Dideoxynucleotides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides / Q000378:metabolism / Q000494:pharmacology
D004272:DNA, Mitochondrial; D054306:Dideoxynucleotides; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human; C017216:2',3'-dideoxythymidine triphosphate; C024157:thymidine 5'-triphosphate