A post-labeling technique for the iodination of DNA damage recognition proteins.
Major advances have recently been made in understanding the nucleotide excision repair pathway in mammalian cells. Although the signaling events responsible for initiating this process are not known, they probably involve proteins, i.e., damage recognition proteins (DRPs), which detect specific types of DNA damage. In this report, we describe a technique for labeling DNA damage recognition proteins. The procedure utilizes iodogen to radio-iodinate proteins bound to DNA modified with the cancer chemotherapy drug, cisplatin. Following iodination, bound proteins are eluted and analyzed on SDS-polyacrylamide gels. We have optimized this procedure such that the labeling reactions are rapid and employ small amounts of 125I. 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 facilitate 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.
Billings
P C
PC
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Cryer
J E
JE
Moy
L Y
LY
Engelsberg
B N
BN
D000818:Animals; D001940:Breast / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002945:Cisplatin / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008970:Molecular Weight
D018736:DNA Adducts; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D007457:Iodine Radioisotopes; C057270:cisplatin-DNA adduct; D002945:Cisplatin