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
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.