Dissection of functional domains of the human DNA replication protein complex replication protein A.
Replication protein A (RPA) is a mammalian single-stranded DNA binding factor essential for DNA replication, repair, and recombination. It is composed of three subunits of 70, 34, and 13 kDa (Rpa1, Rpa2, and Rpa3, respectively). Deletion mapping of the Rpa2 subunit identified the domain required for interaction with Rpa1 and Rpa3 which does not include the N-terminal domain that is phosphorylated during S phase. Deletion mapping of Rpa1 defined three domains. The C-terminal third of the Rpa1 polypeptide binds Rpa2 which itself forms a bridge between Rpa1 and Rpa3. The N-terminal third of Rpa1 bound single-stranded DNA under low stringency conditions only (0.1 M NaCl), while a central domain binds to single-stranded DNA under both low and high stringency conditions (0.5 M NaCl). Binding to p53 requires the N-terminal third of Rpa1 with some contribution from the C-terminal third. The evolutionarily conserved putative zinc finger near the C terminus of Rpa1 was not required for binding to single-stranded DNA, Rpa2, or p53. However, all three subdomains of Rpa1 and the zinc finger were essential for supporting DNA replication in vitro. These experiments are a first step toward defining peptide components responsible for the many functions of the RPA protein complex.
Lin
Y L
YL
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Chen
C
C
Keshav
K F
KF
Winchester
E
E
Dutta
A
A
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D016196:S Phase; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D001616:beta-Galactosidase; D004265:DNA Helicases