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PMID:11927569

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Citation

Bochkareva, E, Korolev, S, Lees-Miller, SP and Bochkarev, A (2002) Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA. EMBO J. 21:1855-63

Abstract

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 ( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A 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.

Links

PubMed PMC125950 Online version:10.1093/emboj/21.7.1855

Keywords

Amino Acid Sequence; Binding Sites; DNA Replication; DNA, Single-Stranded/chemistry; DNA-Binding Proteins/chemistry; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Replication Protein A

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:RFA2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P27694

F

Seeded From UniProt

complete

HUMAN:RFA1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P15927

F

Seeded From UniProt

complete

HUMAN:RFA1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P35244

F

Seeded From UniProt

complete

HUMAN:RFA3

part_of

GO:0005662: DNA replication factor A complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:RFA3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P27694

F

Seeded From UniProt

complete

HUMAN:RFA1

part_of

GO:0005662: DNA replication factor A complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:RFA2

part_of

GO:0005662: DNA replication factor A complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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