A POST-LABELING TECHNIQUE FOR THE IODINATION OF DNA-DAMAGE RECOGNITION PROTEINS

Citation
Pc. Billings et al., A POST-LABELING TECHNIQUE FOR THE IODINATION OF DNA-DAMAGE RECOGNITION PROTEINS, Cancer biochemistry biophysics, 14(4), 1995, pp. 223-230
Citations number
10
Categorie Soggetti
Biology,Oncology,Biophysics
ISSN journal
03057232
Volume
14
Issue
4
Year of publication
1995
Pages
223 - 230
Database
ISI
SICI code
0305-7232(1995)14:4<223:APTFTI>2.0.ZU;2-O
Abstract
Major advances have recently been made in understanding the nucleotide excision repair pathway in mammalian cells. Although the signaling ev ents responsible for initiating this process are not known, they proba bly involve proteins, i.e., damage recognition proteins (DRPs), which detect specific types of DNA damage. In this report, we describe a tec hnique for labeling,DNA damage recognition proteins. The procedure uti lizes iodogen to radio-iodinate proteins bound to DNA modified with th e cancer chemotherapy drug, cisplatin. Following iodination, bound pro teins are eluted and analyzed on SDS-polyacrylamide gels. We have opti mized this procedure such that the labeling reactions are rapid and em ploy small amounts of I-125. Using this procedure, we demonstrate that proteins of 28 and 40 kDa in MCF7 human breast epithelial cells bind to CDDP-DNA. This technique is sensitive and potentially will facilita te the identification of DRPs in samples containing limited amounts of protein, such as small tissue biopsy specimens obtained from patients undergoing diagnostic and/or therapeutic treatment.