Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of -1 2 0 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.[Key Words: SSB; DNA Replication; replication protein A; yeast] Received May 7, 1996; revised version accepted July 24, 1996.As participants in most aspects of DNA metabolism, single-stranded DNA-binding proteins (SSBs) play a crucial role in the control of genetic information. The two cellular SSBs, Escherichia coli SSB (Ecssb) and replication protein A (RPA), bear little structural or amino acid sequence similarity but carry out many of the same essential functions in DNA replication, repair, and recombination. Because many eukaryotic DNA replication proteins show sequence similarity to their homologs in E.
Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDRactivated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D 3 -dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D 3 -induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing. The vitamin D receptor (VDR)1 is a nuclear receptor (NR) family member that mediates the biological actions of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the active hormone of the vitamin D endocrine system. VDR forms a heterodimer with retinoid X receptor (RXR) and binds to specific vitamin Dresponsive elements (VDREs) in the promoter region of target genes to regulate transcription by RNA polymerase II (1, 2). Transcriptional activation through 1,25-(OH) 2 D 3 and VDR is enhanced by nuclear receptor coactivator proteins such as steroid receptor coactivators (SRCs) (3-5) and proteins of the vitamin D receptor-interacting protein (DRIP) complex (6, 7). The SRCs and DRIPs utilize leucine-rich LXXLL motifs (8, 9) to interact in a 1,25-(OH) 2 D 3 -dependent manner with a complementary hydrophobic cleft on the surface of the VDR ligand binding domain. This hydrophobic surface is composed of helices H3, H4, H5, and H12, and it is the ligand-dependent folding of H12 that creates the interaction cleft for SRC binding to NRs (10 -15). The H12 helix of the NRs contains the critical ligand-dependent activation function-2 domain that is essential for ligand activated transcription (16, 17) and ligand-dependent S...
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