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

Journal Article · Thu Jun 01 00:00:00 EDT 1978 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.75.6.2598· OSTI ID:6699362

Smith, C A; Hanawalt, P C

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 (United States)
• OSTI ID: 6699362
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 75:6
• Country of Publication: United States
• Language: English

Related Subjects

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