Abstract
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 heterotrimeric 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. Using a short substrate, such as (dT)12, no stable interaction could be detected with RPA containing inactive domain A (RPA-A
−
) while the K
a
for RPA-B
−
or RPA-C
−
was approximately one third that of wild type RPA. The K
a
of RPA-D
−
was unaffected for substrates 12 to 23 nt in length, but was one third that of wild type RPA for substrates of 40 nt or more. Protein-DNA crosslinking confirms that domain A is essential for RPA to bind substrates of 12 nt or less and that DBD-D (RPA2) requires a minimum of 40 nt to interact with ssDNA. The data support a model in which domain A makes the initial contact with ssDNA, domains A, B, and C (in RPA1) contact substrates up to 23 nt in length, and RPA2 interacts with substrates of 40 - 60 nt.