The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)n folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible M inisatellites (MNs), also called variable number of tandem repeats, are arrays of 5-to 100-bp repeats widely dispersed in eukaryotic genomes (1). Some have been suggested to be implicated in genetic predisposition to various human diseases. Although MNs are known to be hot spots for meiotic recombination in germ cells (1, 2), they are considered to be genetically stable in somatic cells (3). However, they also have been demonstrated to be altered in somatic cells with exposure to chemical carcinogens, ␥ irradiation, or UV irradiation (4-7). They also exhibit alterations in a range of tumors of humans and experimental animals (8-10).The MN Pc-1, located on mouse chromosome 4, consists of tandem repeats of d(GGCAG) (11, 12). The germ-line mutation rate for Pc-1 is 15% per gamete, whereas the mutation rate in somatic cells is 2-3% per 22-24 population doublings (12-14). Many hypervariable MNs, consisting of G-rich repeat units similar to Pc-1, have been found in the mouse and other mammalian genomes, and mouse MNs containing d(GGCAG) n or d(GGCAGG) n motifs are very unstable in germ-line cells (11). Pc-1 and Pc-1-like MNs were demonstrated to be strikingly unstable by DNA fingerprint analysis, both in fibroblasts deficient in DNA-dependent protein kinase activity and in NIH 3T3 cells treated with okadaic acid, an inhibitor of protein phosphatases (13,14).Although the molecular mechanisms underlying the induction of MN mutations are largely unknown, the G-rich strand of Pc-1 is known to form an intramolecular folded-back quadruplex structure under physiological conditions and cause arrest of DNA synthesis (15). We have isolated six MN Pc-1 binding proteins (MNBPs) from NIH 3T3 cells (16). Two of them, MNBP-A and MNBP-B, bind to the G-rich strand of Pc-1, and the other four, MNBP-D, MNBP-E, MNBP-F, and MNBP-G, to the complementary C-rich strand.In this article, we document isolation of cDNA clones encoding MNBP-B and characterization of a recombinant MNBP-B. Sequences of seven proteolytic peptides of purified MNBP-B were determined, and cDNA clones were subsequently isolated. MNBP-B was revealed to be identical to the single-stranded DNA binding protein, UP1 (17), which is a proteolytic product corresponding to the N-terminal 195 aa of the 34-kDa heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (18, 19). DNA binding specificity and in vitro effect on quadruplex structures of UP1 were analyzed to cast light on its biological roles. EMSA. EMSA and analyses of oligonucleotide competition using EMSA were performed as detailed (16) with 32 P-labeled pG8 (final concentration 2 nM)...
Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG) n , the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase. Heterogeneous nuclear ribonucleoprotein (hnRNP)1 D, also known as AUF1, was isolated from a HeLa cell nuclear extract as a protein that binds specifically to single-stranded d(T-TAGGG) n , the human telomeric DNA repeat (1). It also binds to r(UUAGGG) n (1) and the AU-rich element of the 3Ј-untranslated region of mRNA (2). It was shown biochemically that hnRNP D binding to the Gua-rich telomeric strand d(T-TAGGG) n destabilizes intrastrand Gua:Gua pairing and that hnRNP D interacts specifically with telomerase in human cell extracts (3). Thus, the involvement of hnRNP D in the maintenance of telomere DNA has been implied.hnRNP D consists of 306 amino acid residues and comprises two ribonucleoprotein (RNP)-type DNA/RNA-binding domains (BDs), BD1 (70 -173) and BD2 (174 -256), and a region rich in glycine and arginine residues (257-306) (4). The RNP-type BD is one of the most common eukaryotic protein sequence motifs for DNA/RNA binding (5), being found in hundreds of proteins (6 -9). A single BD of hnRNP D, either BD1 or BD2, is able to bind to DNA and RNA in a sequence-specific manner (4, 10, 11). The binding affinity of either BD1 or BD2 is comparable with that of the protein having both BD1 and BD2 (4). These results suggest that the basis of the interactions of hnRNP D with DNA and RNA can be addressed by examination with a single BD. We have already reported the structures of BD1 (10) and BD2 (11). We also qualitatively identified the surfaces of BD1 (10) and BD2 (11) interactive with DNA and RNA on chemical shift perturbation analysis, although it should be kept in mind that the perturbation is caused not only through the direct interaction but also thorough the indirect effect of the interaction. Essentially the same surfaces of the BDs are used for the interactions with DNA and RNA.Here, we present the structure of BD2 complexed with d(...
Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling. A large number of TIR domain structures, including receptors and adaptors, have been determined by X-ray crystallography and NMR. The receptors include TLR1 (9), TLR2 (10), and IL-1R accessory protein-like (IL-1RAPL) (11). Adaptors include myeloid differentiation factor 88 (MyD88) (12) and MyD88 adaptorlike (Mal) (13,14). In addition, AtTIR (15, 16) derived from Arabidopsis thaliana and PdTIR (17) from bacteria have been solved. Each of these TIR domain structures has a ferredoxin fold with five β-strands (βA-βE), five α-helices (αA-αE), and loops connecting β-strands and α-helices (9). Although homotypic interactions of the TIR domains have been proposed based on the crystal structures, most proposed models have small interacting surfaces, possibly due to crystal contacts. Recently, however, a crystal structure of the TLR10 TIR domain was reported that forms a homotypic dimer mediated by the loop connecting βB and αB (designated "BB-loop") (18). Interestingly, BB-loop mutations in TLR4 were reported to be dominant-negative and abrogated downstream signaling (19). TICAM-1 and TICAM-2 harboring BB-loop mutations are also dominant-negative and unable to form homotypic interactions (1, 2), reinforcing the importance of BB-loop-mediated homotypic dimer formation in signal propagation.Despite extensive structural studies, it is not known why homotypic interactions are essential for downstream signaling (20-27). To address this issue, it is necessary to discriminate residues required for homotypic and those required for heterotypic interactions. Here, we first determine the structures of the monomeric BB-loop mutants of the TICAM-1 and TICAM-2 TIR domains using NMR. Then, based on the solution structures of the BB-loop mutants, coupled mutagenesis/yeast two-hybrid experiments, and restrained docking calculations, we show that the homotypic interaction of TICAM-2 TIR is es...
Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.
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