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Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

Journal Article · · Proc. Natl. Acad. Sci. U.S.A.; (United States)
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The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing (/sup 3/H)thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The /sup 3/H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg/sup 2 +/-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.

Research Organization:
Stanford Univ., CA
OSTI ID:
6699362
Journal Information:
Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 75:6; ISSN PNASA
Country of Publication:
United States
Language:
English

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Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560121* -- Radiation Effects on Cells-- External Source-- (-1987)
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL CELLS
BACTERIOPHAGES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CONNECTIVE TISSUE CELLS
DIMERS
DNA
DNA REPLICATION
ELECTROMAGNETIC RADIATION
ENZYMES
FIBROBLASTS
IRRADIATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEASES
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PHOSPHOTRANSFERASES
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
SOMATIC CELLS
STIMULATION
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
VIRUSES
XP CELLS