Replication protein A - Characterization and crystallization of the DNA binding domain

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein in eukaryotic cells. The DNA binding activity of human RPA has been previously localized to the N-terminal 441 amino acids of the 70-kDa subunit, RPA70. We have used a combination of limited proteolysis and mutational analysis to define the smallest soluble fragment of human RPA70 that retains complete DNA binding activity. This fragment comprises residues 181-422. RPA(181-422) bound DNA with the same affinity as the 1-441 fragment and had a DNA binding site of 8 nucleotides or less. RPA70 fragments were subjected to crystal trials in the presence of single-stranded DNA, and diffraction quality crystals were obtained for RPA(181-422) bound to oetadeoxycytidine. The RPA(181-422) co-crystals belonged to the P2(1)2(1)2(1) space group, with unit cell dimensions of a = 34.3 Angstrom, b = 78.0 Angstrom and c = 95.4 Angstrom and diffracted to a resolution of 2.1 Angstrom.