Purification of PCNA as a nucleotide excision repair protein
- 25 May 1992
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 20 (10) , 2441-2446
- https://doi.org/10.1093/nar/20.10.2441
Abstract
Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunobloting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases δ and ε. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.Keywords
This publication has 29 references indexed in Scilit:
- Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.Proceedings of the National Academy of Sciences, 1992
- Synthesis of DNA by DNA polymerase epsilon in vitro.Journal of Biological Chemistry, 1991
- Requirement for the replication protein SSB in human DMA excision repairNature, 1991
- Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication originNature, 1990
- Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (A)BC excision nucleaseBiochemistry, 1990
- Human nucleotide excision repairin vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extractNucleic Acids Research, 1989
- Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extractsCell, 1988
- Increased nuclear cyclin/PCNA antigen staining of non S‐phase transformed human amnion cells engaged in nucleotide excision DNA repairFEBS Letters, 1986
- Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.Proceedings of the National Academy of Sciences, 1985
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976