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Papers In Press, published online ahead of print July 30, 2001
Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854
Corresponding Author: brill{at}mbcl.rutgers.edu
Replication Protein A (RPA), the heterotrimeric SSB of eukaryotes, contains four ssDNA binding domains (DBDs) within its two largest subunits, RPA1 and RPA2. We analyzed the contribution of the four DBDs to ssDNA binding affinity by assaying recombinant yeast RPA in which a single DBD (A, B, C or D) was inactive. Inactivation was accomplished by mutating the two conserved aromatic stacking residues present in each DBD. Mutation of domain A had the most severe effect and eliminated binding to a short substrate such as (dT)12. RPA containing mutations in DBDs B and C bound to substrates (dT)12, 17, and 23 but with reduced affinity compared to wt RPA. Mutation of DBD-D had little or no effect on the binding of RPA to these substrates. However, mutations in domain D did affect the binding to oligonucleotides larger than 23 nt. Protein-DNA crosslinking indicated that DBD-A (in RPA1) is essential for RPA1 to interact efficiently with substrates of 12 nt or less, and that DBD-D (RPA2) interacts efficiently with oligonucleotides of 27 nt or larger. The data support a sequential model of binding in which DBD-A is responsible for the initial interaction with ssDNA, that domains A, B, and C (RPA1) contact 12 to 23 nt of ssDNA, and that DBD-D (RPA2) is needed for RPA to interact with substrates that are 23 to 27 nt in length.
J. Biol. Chem, 10.1074/jbc.M104386200
Submitted on May 14, 2001
Revised on July 24, 2001
Accepted on July 28, 2001
Functional analysis of the four DNA binding domains of Replication protein A: The role of RPA2 in ssDNA binding
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