DNA repair synthesis in human fibroblasts requires DNA polymerase delta
When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone.
- Research Organization:
- Univ. of California, Berkeley
- OSTI ID:
- 5594849
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 263:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
ENZYME ACTIVITY
FIBROBLASTS
DNA REPAIR
GENETIC RADIATION EFFECTS
MONOCLONAL ANTIBODIES
BIOCHEMICAL REACTION KINETICS
ATP
CELL CULTURES
GUANYLIC ACID
HELA CELLS
MAN
ULTRACENTRIFUGATION
ULTRAVIOLET RADIATION
ANIMAL CELLS
ANIMALS
ANTIBODIES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CENTRIFUGATION
CONNECTIVE TISSUE CELLS
ELECTROMAGNETIC RADIATION
ENZYMES
GENETIC EFFECTS
KINETICS
MAMMALS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PRIMATES
RADIATION EFFECTS
RADIATIONS
REACTION KINETICS
RECOVERY
REPAIR
SEPARATION PROCESSES
SOMATIC CELLS
TRANSFERASES
VERTEBRATES
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture
550201 - Biochemistry- Tracer Techniques