Functional analysis of the four DNA binding domains of replication proteinA - The role of RPA2 in ssDNA binding

Replication Protein A (RPA), the heterotrimeric single-stranded DNA (ssDNA) -binding protein of eukaryotes, contains four ssDNA binding domains (DBDs) within its two largest subunits, RPA1 and RPA2. We analyzed the contributio n 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 ac complished by mutating the two conserved aromatic stacking residues present in each DBD. Mutation of domain A had the most severe effect and eliminate d 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 affinit y compared with wild type 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 nucleotides (nt). Pr otein-DNA cross-linking indicated that DBD-A (in RPA1) is essential for RPA 1 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 d ata 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) contac t 12-23 nt of ssDNA, and that DBD-D (RPA2) is needed for RPA to interact wi th substrates that are 23-27 nt in length.