Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface

Iwona M Wyka; Kajari Dhar; Sara K Binz; Marc S Wold

doi:10.1021/bi034930h

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Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface

Journal article

Peer reviewed

Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface

Iwona M Wyka, Kajari Dhar, Sara K Binz and Marc S Wold

Biochemistry (Easton), Vol.42(44), pp.12909-12918

11/11/2003

DOI: 10.1021/bi034930h

PMID: 14596605

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Abstract

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.

Conserved Sequence - genetics

Protein Binding - genetics

Mutagenesis, Site-Directed

Models, Chemical

DNA, Single-Stranded - metabolism

Humans

Protein Structure, Tertiary - genetics

DNA-Binding Proteins - genetics

DNA-Binding Proteins - chemistry

DNA, Single-Stranded - chemistry

Protein Subunits - metabolism

DNA, Single-Stranded - genetics

DNA-Binding Proteins - metabolism

Point Mutation

DNA Mutational Analysis

Surface Properties

Protein Subunits - chemistry

Genetic Vectors

Replication Protein A

Protein Subunits - genetics

Details

Title: Subtitle: Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface
Creators: Iwona M Wyka - Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, Iowa 52242-1109, USA
Kajari Dhar
Sara K Binz
Marc S Wold
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.42(44), pp.12909-12918
Publisher: United States
DOI: 10.1021/bi034930h
PMID: 14596605
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: GM44721 / NIGMS NIH HHS
Language: English
Date published: 11/11/2003
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024419502771

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