| STRUCTURAL-ANALYSIS OF HUMAN REPLICATION PROTEIN-A - MAPPING FUNCTIONAL DOMAINS OF THE 70-KDA SUBUNIT |
|
|
| Author(s): GOMES XV, WOLD MS |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 270 Issue: 9 Pages: 4534-4543 Published: MAR 3 1995 |
| Times Cited: 68 References: 62 |
| 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 do main 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. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV IOWA, SCH MED, DEPT BIOCHEM, IOWA CITY, IA 52242 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: QK084 |
| ISSN: 0021-9258 |