Interactions of mammalian proteins with cisplatin-damaged DNA.
We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.
Turchi
J J
JJ
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA. john.turchi@wright.edu
Henkels
K M
KM
Hermanson
I L
IL
Patrick
S M
SM
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000378:metabolism*; D051716:Replication Protein A; D051760:Xeroderma Pigmentosum Group A Protein
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D011838:Radiation-Sensitizing Agents; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D002945:Cisplatin