Functional domains of the 70-kilodalton subunit of human replication protein A.
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 (RPA 70) 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.
Gomes
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Department of Biochemistry, University of Iowa School of Medicine, Iowa City 52242-1109, USA.
Wold
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MS
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D018741:Replication Origin; D051716:Replication Protein A; D017384:Sequence Deletion; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A