NAR Top Articles - Structural Biology
Structural basis for inhibition of DNA replication by aphidicolin
Baranovskiy, AG; Babayeva, ND; Suwa, Y; Gu, JY; Pavlov, YI; Tahirov, TH
Nucleic Acids Res. 2014, 42, 14013-14021
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Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase alpha (Pol alpha) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol alpha active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol alpha. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives...
Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions
Eldar, A; Rozenberg, H; Diskin-Posner, Y; Rohs, R; Shakked, Z
Nucleic Acids Res. 2013, 41, 8748-8759
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A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson-Crick base pairs associated...
Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments
Lee, W; von Hippel, PH; Marcus, AH
Nucleic Acids Res. 2014, 42, 5967-5977
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DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein-DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer-template DNA constructs labeled with Cy3/Cy5 donor-acceptor Forster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked 'externally' to the bases with flexible tethers, direct 'internal' (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion 'background' signals, thereby simplifying interpretations of both smFRET and smFLD experiments...
Crystal structure of a DNA/Ba2+ G-quadruplex containing a water-mediated C-tetrad
Zhang, DN; Huang, T; Lukeman, PS; Paukstelis, PJ
Nucleic Acids Res. 2014, 42, 13422-13429
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We have determined the 1.50 angstrom A crystal structure of the DNA decamer, d(CCA(CNV)KGCGTGG) (K-CNV, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba2+. The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba2+ ions to occupy adjacent steps in the central ion channel. One ordered Mg2+ facilitates 3'-3' stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the K-CNV nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson-Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation.
Magnetic tweezers measurements of the nanomechanical stability of DNA against denaturation at various conditions of pH and ionic strength
Tempestini, A; Cassina, V; Brogioli, D; Ziano, R; Erba, S; Giovannoni, R; Cerrito, MG; Salerno, D; Mantegazza, F
Nucleic Acids Res. 2013, 41, 2009-2019
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The opening of DNA double strands is extremely relevant to several biological functions, such as replication and transcription or binding of specific proteins. Such opening phenomenon is particularly sensitive to the aqueous solvent conditions in which the DNA molecule is dispersed, as it can be observed by considering the classical dependence of DNA melting temperature on pH and salt concentration. In the present work, we report a single-molecule study of the stability of DNA against denaturation when subjected to changes in solvent. We investigated the appearance of DNA instability under specific external applied force and imposed twist values, which was revealed by an increase in the temporal fluctuations in the DNA extension. These fluctuations occur in the presence of a continuous interval of equilibrium states, ranging from a plectonemic state to a state characterized by denaturation bubbles. In particular, we observe the fluctuations only around a characteristic force value...
A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
Zheng, KW; Wu, RY; He, YD; Xiao, S; Zhang, JY; Liu, JQ; Hao, YH; Tan, Z
Nucleic Acids Res. 2014, 42, 10832-10844
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Human mitochondrial DNA contains a distinctive guanine-rich motif denoted conserved sequence block II (CSB II) that stops RNA transcription, producing prematurely terminated transcripts to prime mitochondrial DNA replication. Recently, we reported a general phenomenon that DNA: RNA hybrid G-quadruplexes (HQs) readily form during transcription when the non-template DNA strand is guanine-rich and such HQs in turn regulate transcription. In this work, we show that transcription of mitochondrial DNA leads to the formation of a stable HQ or alternatively an unstable intramolecular DNA G-quadruplex (DQ) at the CSB II. The HQ is the dominant species and contributes to the majority of the premature transcription termination. Manipulating the stability of the DQ has little effect on the termination even in the absence of HQ; however, abolishing the formation of HQs by preventing the participation of either DNA or RNA abolishes the vast majority of the termination. These results demonstrate that the type of G-quadruplexes (HQ or DQ) is a crucial determinant in directing the transcription termination at the CSB II...
Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats
Malgowska, M; Gudanis, D; Kierzek, R; Wyszko, E; Gabelica, V; Gdaniec, Z
Nucleic Acids Res. 2014, 42, 10196-10207
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Trinucleotide repeats are microsatellite sequences that are polymorphic in length. Their expansion in specific genes underlies a number of neurodegenerative disorders. Using ultraviolet-visible, circular dichroism, nuclear magnetic resonance (NMR) spectroscopies and electrospray ionization mass spectrometry, the structural preferences of RNA molecules composed of two and four repeats of AGG, CGG and UGG in the presence of K+, Na+ and NH4+ were analysed. (AGG)(2)A, (AGG)(4)A, p(UGG)(2)U and p(UGG)(4)U strongly prefer folding into G-quadruplexes, whereas CGG-containing sequences can adopt different types of structure depending on the cation and on the number of repeats. In particular, the two-repeat CGG sequence folds into a G-quadruplex in potassium buffer. We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)(2)A, whereas mixed G: C: G: C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)(2)C and p(UGG)(2)U, respectively. Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding...
Structural insights into Paf1 complex assembly and histone binding
Chu, XL; Qin, XH; Xu, HS; Li, L; Wang, Z; Li, FZ; Xie, XQ; Zhou, H; Shen, YQ; Long, JF
Nucleic Acids Res. 2013, 41, 10619-10629
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The highly conserved Paf1 complex (PAF1C) plays critical roles in RNA polymerase II transcription elongation and in the regulation of histone modifications. It has also been implicated in other diverse cellular activities, including posttranscriptional events, embryonic development and cell survival and maintenance of embryonic stem cell identity. Here, we report the structure of the human Paf1/Leo1 subcomplex within PAF1C. The overall structure reveals that the Paf1 and Leo1 subunits form a tightly associated heterodimer through antiparallel beta-sheet interactions. Detailed biochemical experiments indicate that Leo1 binds to PAF1C through Paf1 and that the Ctr9 subunit is the key scaffold protein in assembling PAF1C. Furthermore, we show that the Paf1/Leo1 heterodimer is necessary for its binding to histone H3, the histone octamer, and nucleosome in vitro. Our results shed light on the PAF1C assembly process and substrate recognition during various PAF1C-coordinated histone modifications.
On the structural basis and design guidelines for type II topoisomerase-targeting anticancer drugs
Wu, CC; Li, YC; Wang, YR; Li, TK; Chan, NL
Nucleic Acids Res. 2013, 41, 10630-10640
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Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme-DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 beta-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3'-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex...
Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1
Montemayor, EJ; Katolik, A; Clark, NE; Taylor, AB; Schuermann, JP; Combs, DJ; Johnsson, R; Holloway, SP; Stevens, SW; Damha, MJ; Hart, PJ
Nucleic Acids Res. 2014, 42, 10845-10855
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The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2',5'-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2',5'-phosphodiester recognition and explain why the enzyme lacks activity toward 3',5'-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents...
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