Structural Characterization of Human Rpa Sequential Binding to Single-stranded Dna Using Ssdna As a Molecular Ruler.
From: Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Biochemistry
- Publish Date: Jul 2007
- ISSN: 0006-2960
- Volume: 46
- Issue: 28
- Pages: 8226-33
- Medium: Print
- Language: English
- Citation (JAMA): Cai Lifeng, Roginskaya Marina, Qu Youxing, et al. Structural Characterization of Human Rpa Sequential Binding to Single-stranded Dna Using Ssdna As a Molecular Ruler.. Biochemistry Jul 2007;46:8226-33
Abstract
Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5’-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt ( approximately 19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt ( approximately 34 A). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed.
Mesh Headings (Keywords): Binding Sites, DNA, Single-Stranded, Humans, Models, Molecular, Mutation, Protein Binding, Protein Structure, Tertiary, Replication Protein A, Spectrometry, Fluorescence
Check for Full Text / PubMed Unique Identifier (PMID): 17583916
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