A role for the Saccharomyces cerevisiae Rtt109 histone acetyltransferase in R-loop homeostasis and associated genome instability

The stability of the genome is occasionally challenged by the formation of DNA–RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA–RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On the one hand, Rtt109 prevents DNA–RNA hybridization by the acetylation of histone H3 lysines 14 and 23 and, on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA–RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability.     

BRCA2 promotes DNA‐RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair

The BRCA2 tumor suppressor is a DNA double‐strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2‐deficient cells spontaneously accumulate DNA‐RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD‐box RNA helicase DDX5 as a BRCA2‐interacting protein. DDX5 associates with DNA‐RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA‐RNA hybrid‐unwinding activity of DDX5 helicase. An impaired BRCA2‐DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2‐T207A, reduces the association of DDX5 with DNA‐RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA‐RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.     

Thioguanine substitution alters DNA cleavage mediated by topoisomerase II.

Thiopurines and topoisomerase II-targeted drugs (e.g., etoposide) are widely used anticancer drugs. However, topoisomerase II-targeted drugs can cause acute myeloid leukemia, with the risk of this secondary leukemia linked to a genetic defect in thiopurine catabolism. Chronic thiopurines result in thioguanine substitution in DNA. The effect of these substitutions on DNA topoisomerase II activity is not known. Our goal was to determine whether deoxythioguanosine substitution alters DNA cleavage stabilized by human topoisomerase II. We studied four variations of a 40 mer oligonucleotide with a topoisomerase II cleavage site, each with a single deoxythioguanosine in a different position relative to the cleavage site (-1 or +2 in the top and +2 or +4 in the bottom strand). Deoxythioguanosine substitution caused position-dependent quantitative effects on cleavage. With the -1 or +2 top and +2 or +4 bottom substitutions, mean topoisomerase II-induced cleavage was 0.6-, 2.0-, 1.1-, and 3.3-fold that with the wild-type substrate (P=0. 011, < 0.008, 0.51, and < 0.001, respectively). In the presence of 100 microM etoposide, cleavage was enhanced for wild-type and all thioguanosine-modified substrates relative to no etoposide, with the +4 bottom substitution showing greater etoposide-induced cleavage than the wild-type substrate (P=0.015). We conclude that thioguanine incorporation alters the DNA cleavage induced by topoisomerase II in the presence and absence of etoposide, providing new insights to the mechanism of thiopurine effect and on the leukemogenesis of thiopurines, with or without topoisomerase inhibitors.     

Inferring primase-DNA specific recognition using a data driven approach

DNA–protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA–protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA–protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.     

DNA Relatedness,Divergence, and Sibling Species of the Lactic Acid Bacterium Streptococcus thermophilus

Previously, five distinct groups with 80–90% intragroup DNA homology values were revealed among 19 lactic acid–producing bacterial strains. The study of 39 new strains of thermophilic streptococci in the present work allowed us to reveal the sixth DNA homology group. The nine strains of this group are close, at 55–70% DNA homology levels, to the type strain Streptococcus thermophilus ATCC 19258. Group VI showed a low level of DNA–DNA reassociation (20–30%) with the DNA homology groups I, II, III, and V. The intergroup DNA–DNA reassociation values determined from DNA renaturation rates varied from 20 to 50%. These data were interpreted as indicative of the existence of at least four sibling species among the thermophilic streptococci studied.     

A simple fluorimetric method for the estimation of DNA–DNA relatedness between closely related microorganisms by thermal denaturation temperatures

Determination of whole-genome DNA–DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA–DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA–DNA relatedness between closely related species.     

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.     

Receptor-Mediated Transfer of DNA–Galactosylated Poly-L-lysine Complexes into Mammalian Cells in vitro and in vivo

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA–poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA–poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA–poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.     

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.     

Structure of DNA Complexes with Nonhistone Chromosomal Protein HMGB1 in the Presence of Manganese Ions

The complexes of DNA–HMGB1 protein–manganese ions have been studied using the circular dichroism (CD) technique. It was shown that the interactions of both the protein and metal ions with DNA in this three-component system differ from those in two-component complexes. The manganese ions did not affect the CD spectrum of the free HMGB1 protein. However, Mn²⁺ ions induced considerable changes in the CD spectrum of free DNA in the spectral range of 260–290 nm. The presence of Mn²⁺ ions prevented the formation of the ordered supramolecular structures typical of HMGB1–DNA complexes. The interaction of manganese ions with DNA had a marked influence on the local DNA structure, changing the properties of protein-binding sites and resulting in a marked decrease in cooperativity of HMGB1–DNA binding. Such changes in the mode of protein–DNA interactions occurred at concentrations as small as 0.01 mM Mn²⁺. Moreover, the changes in local DNA structure induced by the manganese ions promoted the appearance of new HMGB1 binding sites in the DNA double helix. At the same time, interactions with the HMGB1 protein induced alterations in the structure of the DNA double helix, which increased with an increase in the protein-to-DNA ratio. These alterations made the DNA–protein complex especially sensitive to manganese ions. Under these conditions the Mn²⁺ ions strongly affected the DNA structure, which was reflected in abrupt changes in the CD spectra of DNA in the complex in the range of 260–290 nm. Thus, structural changes in the DNA double helix in three-component DNA–HMGB1–Mn²⁺ complexes result from the combined and interdependent interactions of DNA with Mn²⁺ ions and HMGB1 molecules.     

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2′-fluoro, 2′-O-propyl, 2′-O-methoxyethyl and 2′-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA–DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3°C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA–RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0°C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.     

Ultrasensitive isolation,identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA–protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA–protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA–protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA–protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1–DNA and Top2a–DNA adducts in human cells, and gyrase–DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine.     

Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

Design and preparation of layered composite materials alternating between nucleic acids and proteins has been elusive due to limitations in occurrence and geometry of interaction sites in natural biomolecules. We report the design and kinetically controlled stepwise synthesis of a nano-sandwich composite by programmed noncovalent association of protein, DNA and RNA modules. A homo-tetramer protein core was introduced to control the self-assembly and precise positioning of two RNA–DNA hybrid nanotriangles in a co-parallel sandwich arrangement. Kinetically favored self-assembly of the circularly closed nanostructures at the protein was driven by the intrinsic fast folding ability of RNA corner modules which were added to precursor complex of DNA bound to the protein. The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies. The synthesis strategy for the nano-sandwich composite provides a general blueprint for controlled noncovalent assembly of complex supramolecular architectures from protein, DNA and RNA components, which expand the design repertoire for bottom-up preparation of layered biomaterials.     

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.     

Dimeric assembly of human Suv3 helicase promotes its RNA unwinding function in mitochondrial RNA degradosome for RNA decay

Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA–DNA, DNA–RNA, and RNA–RNA duplexes with a long 3′ overhang (≥10 nucleotides). The C‐terminal tail (CTT)‐truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo‐form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase–Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell‐shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.     

Formation of DNA Complexes with Actinomycins in Aqueous Solutions and Films

The mechanisms of DNA interaction with actinomycin D (AMD), 7-amino-actinomycin D (7-AAMD), and ethidium bromide (EtBr) were studied in aqueous solutions and in the condensed state (films coating plates). The use of the methods of absorption (UV, IR, and visible spectral ranges) and fluorescence (steady-state, polarization, and phase-modulation) spectroscopy revealed that (1) the formation of DNA complexes with 7-AAMD in solution was not accompanied by energy transfer from photoexcited nucleotides to phenoxazone chromophore and (2) the mechanism of ligand incorporation was distinct from stacking. In the film of the DNA–7-AAMD complex, which simulated the native state in a biological cell, the energy transfer efficiency was high. This indicates that a stacking-type mechanism underlies actinomycin intercalation into DNA. In the presence of high concentrations of 7-AAMD in the film, DNA denatured and its double-helical structure degraded. In the DNA–AMD complex, the native B-form of DNA molecule was conserved both in films and in solution.