Presence Of Ribonucleotides Research Articles

Ribonucleotide Incorporation by Eukaryotic B-Family Replicases and Its Implications for Genome Stability.

Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.

Rotational and translational positions determine the structural and dynamic impact of a single ribonucleotide incorporated in the nucleosome

Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, causing replication stress, chromosome fragility, gross chromosomal rearrangements, and other mutagenic events. Furthermore, nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. Notably, nucleosome formation is significantly reduced by a single ribonucleotide. Single ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme, which incises the DNA backbone on the 5′-side of the ribonucleotide. While the structural implications of a single ribonucleotide in free duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the possible consequent impact on RER, have not been explored. We have carried out 3.5 μs molecular dynamics simulations of a single ribonucleotide incorporated at various translational and rotational positions in a nucleosome core particle. We find that the presence of the 2′−OH group on the ribose impacts the local conformation and dynamics of both the ribonucleotide and nearby DNA nucleotides as well as their interactions with histones; the nature of these disturbances depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. The ribonucleotide’s preferred C3′-endo pucker is stabilized by interactions with the histones, and furthermore the ribonucleotide can cause dynamic local duplex disturbance involving an abnormal C3′-endo population of the adjacent deoxyribose pucker, minor groove opening, ruptured Watson-Crick pairing, and duplex unwinding that are governed by translation-dependent histone-nucleotide interactions. Possible effects of these disturbances on RER are considered.

Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells.

A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5' end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2'-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks. Using EdUseq-HU, the spatiotemporal program of DNA replication in human cell lines can be interrogated in <2 weeks. The protocol requires basic cell culture and molecular biology skills, as well as familiarity with the Perl programming language and the Linux operating system.

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Translesion synthesis DNA polymerase \u03b7 exhibits a specific RNA extension activity and a transcription-associated function

Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Polη performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 103 and 102–fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Polη, and that Polη is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Polη. In summary, these results suggest that the new RNA synthetic activity of Polη can have in vivo relevance.

Processing ribonucleotides incorporated during eukaryotic DNA replication.

The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease.

Ribonucleotide triggered DNA damage and RNA-DNA damage responses

Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

SummaryThe presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ɛ) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ɛ copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ɛ were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ɛ, by inactivating the 3′-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.

Specific discontinuities in Leishmania tarentolae minicircles map within universally conserved sequence blocks

Specific discontinuities remaining in the daughter strands of newly synthesized minicircles from Crithidia fasciculata and Trypanosoma equiperdum have been shown to lie within conserved sequence block (CSBs) present in minicircles from all species of trypanosomes. The presence of ribonucleotides at the 5′ ends of the L-strand in both cases suggests that the conserved sequences may constitute a replication origin. However, specific discontinuities have only been mapped in organisms having unusually homogeneous populations of minicircles. The specific nicks in minicircles from the heterogeneous population present in Leishmania tarentolae have now been mapped to predicted sites within the CSBs. These results are consistent with a universal mechanism of minicircle replication dictated by a conserve sequence motif present both in minicircles having a high degree of sequence heterogeneity as well as in relatively homogeneous populations.

Both strands of polyoma DNA are replicated discontinuously with ribonucleotide primers in vivo.

Nascent polyoma DNA molecules were isolated after pulse-labeling of infected murine 3T6 cells with [3H]thymidine. The extent of digestion of these DNA molecules by spleen exonuclease was increased by exposure to alkali or RNase, suggesting that ribonucleotides were present at or near the 5' terminal of the newly synthesized pieces of DNA. Intermediates shorter than 300 nucleotides were hybridized to the separated strands of restriction enzyme fragments of the polyoma genome: 2.5 to 3-fold more radioactivity was found in the strand whose synthesis is necessarily discontinuous (the lagging strand) than in the strand whose synthesis is potentially continuous (the leading strand) than in the strand whose synthesis is potentially continuous (the leading strand). Separation of the strands of [5'-32P]DNA molecules showed that the excess [3H]thymidine in lagging-strand molecules was not simply the result of an increased number of molecules. Therefore, assuming equivalent efficiencies of labeling, lagging-strand pieces must be slightly longer than those with leading-strand polarity. The presence of ribonucleotides on the 5' termini of molecules with both leading- and lagging-strand polarity was demonstrated by (i) release of 32P-ribonucleoside diphosphates upon alkaline hydrolysis of [5'-32P]DNA separated according to replication polarity and (ii) the change in the degree of self-annealing of nascent molecules upon preferential degradation of DNA molecules possessing initiator RNA moieties by spleen exonuclease. We conclude that replication of polyoma DNA in vivo occurs discontinuously on both sides of the growing fork, using RNA as the major priming mechanism.

Histochemistry of the keratohyalin granules in human oral leukoplakia.

The keratohyalin granules from 25 human oral leukoplakias, showing benign hyperorthokeratosis histologically, were examined employing a series of histochemical techniques. The tissues were fixed in 10% neutral buffered formalin, 80% methanol, or Carnoy's fluid. The keratohyalin granules stained intensely with Pauly's reagent, Congo red and Harris hematoxylin, indicating the presence of proteins. This was confirmed by abolishing the staining reaction by pretreatment with proteolytic enzymes. The keratohyalin granules also reacted with methyl green-pyronin by staining pink at their peripheries; this staining was abolished by pretreatment with ribonuclease, indicating the presence of ribonucleotides. The keratohyalin granules partially stained with toluidine blue and colloidal iron, indicating the presence of acid polysaccharides. The keratohyalin granules did not react with the Feulgen reagent, suggesting the absence of DNA. Our studies indicate that the keratohyalin granules in human oral leukoplakia are primarily protein(s) complexed with polyribonucleotides. The presence of a carbohydrate moiety suggests the possibility of a protein-polysaccharide component in the granules.

The presence of ribonucleotides in mature closed-circular mitochondrial DNA.

Mitochondrial DNA from mouse and HeLa cells is nicked by alkali and chick-embryo ribonuclease H. It has been concluded that ribonucleotides are present in the closed-circular duplex. A comparative study of the scission rates at pH 13.0 of RNA and of mitochondrial DNAs indicates that there are no more than 10 ribonucleotides in the major fraction of the mitochondrial DNAs.

Adaptation of mammalian cells to cold: VII. Molecular basis of cold resistance in LC 2 cells

LC 2 cells characterized by a higher cold-resistance in comparison with the cells of parental wild population possess higher 3H-uridine pool, the difference being especially evident between the lipidic extracts. Higher metabolic pool is connected in LC 2 cells with a greater RNA stability and determines higher cold-resistance of cells. The number of surviving unadapted L cells in the presence of ribonucleotides in the medium after 4 weeks at 4 °C is identical with the number of surviving LC 2 cells in control medium.