Key Concepts:

Figure 1. Structure of RPA. (a) Structural domains of the human RPA subunits. RPA is expressed as a heterotrimer consisting of either the RPA1, RPA2 and RPA3 subunits (canonical RPA) or the RPA1, RPA4 and RPA3 subunits (alternative RPA). RPA1 consists of: DBD-F, which mediates interactions with other protein and regulates RPA functions; DBD-A and DBD-B which comprise the high-affinity DNA-binding core and DBD-C which is required for subunit interactions and contains four-conserved cysteine residues required for coordinating the Zn²⁺ ion. RPA2 and the homologous RPA4 subunit consist of a central DNA-binding domain (DBD-D or DBD-G, respectively) flanked by a C-terminal winged helix domain. RPA2, and putatively RPA4 also encode an N-terminal phosphorylation domain (denoted ‘P’). RPA2 is to phosphorylate during the cell cycle and follow DNA damage and regulate RPA function (also see Figure 3). RPA3 consists of DBD-E and is required for stable assembly of the complex. Flexible, unstructured linker regions are shown as faded rectangles. (b) Hypothetical composite model of canonical RPA. Ribbon structures for each of the domains of RPA and linker regions are shown to scale in an arbitrary conformation. Zinc ion associated with DBD-C is shown as a black circle.

Figure 2. Structure of RPA high-affinity DNA-binding domains. (a) The structure of the high-affinity DNA-binding domain of human RPA1 (residues 181–422) bound to single-stranded DNA. Ribbon diagram with RPA coloured in blue (-strands), purple ( helices), orange (random coil) and the DNA coloured in red. Amino acid numbers are shown in white. (b) View down the axis of the DNA-binding channel. Modified from Bochkarev et al. (1997).

Figure 3. Schematic representation of RPA function in different cell processes. Known protein–protein interactions and protein complexes are diagrammed. Overlapping of RPA (purple oval) with other proteins indicates specific interaction with that protein. The orientations and stoichiometry of complexes have been simplified. (a) RPA in DNA replication. Eukaryotic replication fork, showing protein interactions important for coordination of leading and lagging strand synthesis. RPA interactions with the DNA template, DNA polymerase alpha/primase and cellular helicase (e.g. MCM 2–7 complex) are thought to be important for efficient DNA synthesis. The lagging strand is synthesized as a series of short fragments called Okazaki fragments (red DNA strand with zig-zag RNA primers), and are also bound by RPA. (b) Recombination is used in cells to repair double-strand breaks in the DNA backbone and involves exchange of genetic material between the damaged DNA strand and a similar region of DNA on the sister chromosome. This process is initiated by RAD51 and RAD52 binding to RPA. (c) RPA in DNA repair. RPA is involved in multiple repair pathways (nucleotide excision repair shown). RPA functions both in recognition of damaged DNA and the recruitment of many proteins to the DNA lesion, such as XPA and XPG. (d) Interactions with transcription factors and oncogenes. RPA interacts with many transcription factors and can regulate their activity, and impact gene expression. RPA may also regulate gene transcription through interactions with chromatin remodelling proteins such as menin. (e) RPA in DNA damage response signalling. RPA is one of several key proteins that initiates the activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair, senescence or apoptosis. Following DNA damage, RPA can interact with the sensor complexes MRE11/NBS1/RAD50 and RAD9/RAD1/HUS1/RAD17 to initiate signalling. ATM and ATR kinases are subsequently recruited and phosphorylate several proteins to activate the DNA damage checkpoint (red arrows), including the RPA2 subunit.