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Structural Analysis of Human Replication Protein A: MAPPING FUNCTIONAL DOMAINS OF THE 70-kDa SUBUNIT
Journal article   Open access   Peer reviewed

Structural Analysis of Human Replication Protein A: MAPPING FUNCTIONAL DOMAINS OF THE 70-kDa SUBUNIT

Xavier V Gomes and Marc S Wold
The Journal of biological chemistry, Vol.270(9), pp.4534-4543
03/03/1995
DOI: 10.1074/jbc.270.9.4534
PMID: 7876222
url
https://doi.org/10.1074/jbc.270.9.4534View
Published (Version of record) Open Access

Abstract

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.

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