Abstract
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.
Highlights
RPA1 is absolutely required in the in vitro SV40-based DNA replication reaction [1,2,3] and is important for many other DNA-mediated processes
These results provide a functional map of the Replication protein A (RPA) complex and suggest that, beside the binding of single-stranded DNA and the recruitment of Rpa2 and 3 to the replication apparatus, the Rpa1 subunit executes additional functions essential for DNA replication
When pJGRPA2 was transformed into the yeast (EGY40::pSH18 –34, pEGRPA1), and the expression of the JgRpa2 fusion protein induced with galactose, significant -galactosidase activity was produced (Fig. 1)
Summary
RPA1 is absolutely required in the in vitro SV40-based DNA replication reaction [1,2,3] and is important for many other DNA-mediated processes. In addition RPA associates with proteins containing acidic transcriptional activator domains such as p53, VP16, and the DNA repair protein XP-G These interactions have been proposed to inhibit DNA binding by RPA and to recruit RPA for replication and repair [13,14,15,16]. The third uses recombinant RPA holocomplexes to confirm the findings from the earlier assays and to determine the domains of Rpa essential for SV40-based DNA replication Together, these results provide a functional map of the RPA complex and suggest that, beside the binding of single-stranded DNA and the recruitment of Rpa and 3 to the replication apparatus, the Rpa subunit executes additional functions essential for DNA replication
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