Nucleic Acids Research, Vol 27, Issue 15 3183-3189, Copyright © 1999 by Oxford University Press
U Schweizer, T Hey, G Lipps and G Krauss
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.
ARTICLES
Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA
Lehrstuhl fur Biochemie, Universitat Bayreuth, Universitatsstrasse 30, D-95447 Bayreuth, Germany.
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