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. RPA181-422 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 RPA181-422 bound to octadeoxycytidine. The RPA181-422 co-crystals belonged to the P2(1)2(1)2(1) space group, with unit cell dimensions of a = 34.3 A, b = 78.0 A, and c = 95.4 A and diffracted to a resolution of 2.1 A.
Pfuetzner
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RA
Cancer Research Group, Institute for Molecular Biology and Biotechnology, Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Bochkarev
A
A
Frappier
L
L
Edwards
A M
AM
D018360:Crystallography, X-Ray; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins; D051716:Replication Protein A
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A