Multiple thymine dimer excising nuclease activities in extracts of human KB cells

• Published in: Biochemistry (Easton) 1978-03, Vol.17 (5), p.850-857
• Main Authors: Cook, Kem H, Friedberg, Errol C
• Format: Article
• Language: English
• Subjects: Cations, Divalent - pharmacology; Cell Line; Centrifugation, Density Gradient; Deoxyribonucleases - isolation & purification; Deoxyribonucleases - metabolism; DNA Repair; Endonucleases - isolation & purification; Endonucleases - metabolism; Humans; Isoelectric Focusing; Molecular Weight; Pyrimidine Dimers - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00598a016

Crude extracts of human KB cells grown in suspension culture contain enzyme activity that catalyzes the preferential excision of thymine-containing pyrimidine dimers from UV-irradiated E. coli DNA specifically incised adjacent to dimer sites. Fractionation of KB cell crude extracts reveals the presence of three such activities with distinct affinities for both DEAE-cellulose and phosphocellulose. One of the activities (activity B) is distinguished by its s 20,w (2.6) and isoelectric point (9.0) from the other two (activities A and C) which have similar s 20,w's (3.0-3.2) and isoelectric points (6.0). All three differ in their extent of stimulation by divalent cation and inhibition by NaCl or a sulfhydryl group inhibitor. These results indicate that multiple 5' leads to 3' dimer excision nuclease activities exist in human cells; however, there is as yet no direct evidence that these enzymes are functional in nucleotide excision repair in vivo.