In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

• Published in: Biochemistry (Easton) 1988-09, Vol.27 (19), p.7247-7254
• Main Authors: Dresler, Steven L, Frattini, Mark G, Robinson-Hill, Rona M
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture; ANIMAL CELLS; ANIMALS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BIOLOGICAL EFFECTS; BIOLOGICAL RADIATION EFFECTS; BIOLOGICAL RECOVERY; BIOLOGICAL REPAIR; CARBON 14 COMPOUNDS; Cell Line; Cell Membrane Permeability; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; Deoxycytosine Nucleotides - metabolism; Deoxyguanine Nucleotides - metabolism; Deoxyribonucleotides - metabolism; DNA - biosynthesis; DNA Polymerase II - metabolism; DNA Polymerase III; DNA POLYMERASES; DNA REPAIR; DNA Repair - radiation effects; DNA REPLICATION; DNA-Directed DNA Polymerase - metabolism; ELECTROMAGNETIC RADIATION; ENZYMES; EXTREME ULTRAVIOLET RADIATION; FIBROBLASTS; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Humans; ISOTOPES; KINETICS; LABELLED COMPOUNDS; LIGHT NUCLEI; MAMMALS; MAN; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; NUCLEI; NUCLEIC ACID REPLICATION; NUCLEOTIDYLTRANSFERASES; ODD-ODD NUCLEI; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; Potassium Chloride - pharmacology; PRIMATES; RADIATION EFFECTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADIATIONS; RADIOINDUCTION; RADIOISOTOPES; REACTION KINETICS; RECOVERY; REPAIR; SOMATIC CELLS; Thymine Nucleotides - metabolism; TRANSFERASES; ULTRAVIOLET RADIATION; Ultraviolet Rays; VERTEBRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00419a011

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.