Functional Domains of the 70-Kilodalton Subunit of Human Replication Protein A
- 1 January 1996
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 35 (32), 10558-10568
- https://doi.org/10.1021/bi9607517
Abstract
Human replication protein A (RPA) is a single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. This heterotrimeric complex is required for multiple processes in DNA metabolism including DNA replication, DNA repair, and recombination. Previous studies have suggested that the 616 amino acid, 70-kDa subunit of RPA (RPA70) is composed of multiple structural/functional domains. We used a series of N-terminal deletions of RPA70 to define the boundaries of these domains and elucidate their functions. Mutant RPA complexes missing residues 1−168 of RPA70 bound ssDNA with high affinity and supported SV40 replication in vitro. In contrast, deletions extending beyond residue 168 showed a decreased affinity for ssDNA and were inactive in SV40 DNA replication. When residues 1−381 were deleted, the resulting truncated RPA70 was unable to bind ssDNA but still formed a stable complex with the 32- and 14-kDa subunits of RPA. Thus, the C-terminal domain of RPA70 is both necessary and sufficient for RPA complex formation. These data indicate that RPA70 is composed of three functional domains: an N-terminal domain that is not required for ssDNA binding or SV40 replication, a central DNA-binding domain, and a C-terminal domain that is essential for subunit interactions. For all mutant complexes examined, both phosphorylation of the 32-kDa subunit of RPA and the ability to support T antigen-dependent, origin-dependent DNA unwinding correlated with ssDNA binding activity.Keywords
This publication has 12 references indexed in Scilit:
- Structural Analysis of Human Replication Protein AJournal of Biological Chemistry, 1995
- Inhibition of DNA replication factor RPA by p53Nature, 1993
- Single‐stranded DNA binding protein from calf thymusEuropean Journal of Biochemistry, 1992
- Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.The Journal of cell biology, 1992
- Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding proteinJournal of Molecular Biology, 1992
- DNA unwinding activity of replication protein AFEBS Letters, 1992
- Phosphorylation of Replication Protein A: A Role for cdc2 Kinase in G1/S RegulationCold Spring Harbor Symposia on Quantitative Biology, 1991
- Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells.Genes & Development, 1990
- [7] High-level translation initiationMethods in enzymology, 1990
- Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replicationNature, 1989