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Structure of the RPA trimerization core and its role in the multistep DNA‐binding mechanism of RPA
Author(s) -
Bochkareva Elena,
Korolev Sergey,
LeesMiller Susan P.,
Bochkarev Alexey
Publication year - 2002
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/21.7.1855
Subject(s) - oligonucleotide , dna , replication protein a , protein subunit , dna binding domain , chemistry , binding domain , helix (gastropod) , hmg box , conformational change , binding site , biophysics , stereochemistry , crystallography , biology , dna binding protein , biochemistry , transcription factor , ecology , snail , gene
The human single‐stranded DNA‐binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8–10 nucleotides (nt)] and stable (∼30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 Å structure of the RPA trimerization core comprising the C‐terminal DNA‐binding domain of subunit RPA70 (DBD‐C), the central DNA‐binding domain of subunit RPA32 (DBD‐D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)‐fold and flanked by a helix at the C‐terminus. Trimerization is mediated by three C‐terminal helices arranged in parallel. The OB‐fold of DBD‐C possesses unique structural features; embedded zinc ribbon and helix–turn–helix motifs. Using time‐resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8–10 to 30 nt mode is mediated by DNA binding with the trimerization core.