Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA.
The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of damaged DNA sites during nucleotide excision repair. Detailed structural information on the binding of these proteins to DNA lesions is however lacking. We have studied the binding of human RPA (hRPA) and hRPA-XPA-complexes to model oligonucleo-tides containing a single 1, 3-d(GTG)-cisplatin-modification by photocrosslinking and electrophoretic mobility shift experiments. The 70 kDa subunit of hRPA can be crosslinked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo-2"-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crosslinking is dependent on the presence of the DNA lesion and occurs preferentially at its 5"-side. Examination of the crosslinking efficiency in dependence on the position of the 5-IdU chromophore indicates a specific positioning of hRPA with respect to the platination site. When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA. Our mobility shift experiments directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA. The affinity of the XPA-hRPA complex to the damaged DNA is increased by more than one order of magnitude as compared to hRPA alone.
Schweizer
U
U
Lehrstuhl für Biochemie, Universität Bayreuth, Universitätsstrasse 30, D-95447 Bayreuth, Germany.
Hey
T
T
Lipps
G
G
Krauss
G
G
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007065:Idoxuridine / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D017671:Platinum Compounds / Q000378:metabolism; D011487:Protein Conformation; D051716:Replication Protein A; D012965:Sodium Chloride / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D017671:Platinum Compounds; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D012965:Sodium Chloride; D002918:Chymotrypsin; D014357:Trypsin; D007065:Idoxuridine; D002945:Cisplatin