Structural analysis of human replication protein A. Mapping functional domains of the 70-kDa subunit.
Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is essential for DNA metabolism. Human RPA is composed of subunits of 70, 32, and 14 kDa with intrinsic DNA-binding activity localized to the 616-amino acid, 70-kDa subunit (RPA70). We have made a series of C-terminal deletions to map the functional domains of RPA70. Deletion of the C terminus resulted in polypeptides that were significantly more soluble than RPA70 but were unable to form stable complexes with the other two subunits of RPA. These data suggest that the C-terminal region of RPA70 may be important for complex formation. The DNA-binding domain was localized to a region of RPA70 between residues 1 and 441. A mutant containing residues 1-441 bound oligonucleotides with an intrinsic affinity close to wild-type RPA complex. This mutant also appeared to bind with reduced cooperativity. We conclude that the C terminus of RPA70 and the 32- and 14-kDa subunits are not involved directly with interactions with DNA but may have a role in cooperativity of RPA binding. RPA70 deletion mutants were not able to support DNA replication even in the presence of a complex of the 32- and 14-kDa subunits, suggesting that the heterotrimeric complex is essential for DNA replication. The putative zinc finger in the C terminus of RPA70 is not required for single-stranded DNA-binding activity.
Gomes
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Department of Biochemistry, University of Iowa School of Medicine, Iowa City 52242-1109.
Wold
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MS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D051716:Replication Protein A; D017384:Sequence Deletion
D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA