Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals

Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1^eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.     

Structural Impact of Single Ribonucleotide Residues in DNA

Single ribonucleotide intrusions represent the most common nonstandard nucleotide type found incorporated in genomic DNA, yet little is known of their structural impact. This lesion incurs genomic instability in addition to affecting the physical properties of the DNA. To probe for structural and dynamic effects of single ribonucleotides in various sequence contexts—AxC, CxG, and GxC, where x=rG or dG—we report the structures of three single‐ribonucleotide‐containing DNA duplexes and the corresponding DNA controls. The lesion subtly and locally perturbs the structure asymmetrically on the 3′ side of the lesion in both the riboguanosine‐containing and the complementary strand of the duplex. The perturbations are mainly restricted to the sugar and phosphodiester backbone. The ribose and 3′‐downstream deoxyribose units are predominately in N‐type conformation; backbone torsion angles ? and/or ζ of the ribonucleotide or upstream deoxyribonucleotide are affected. Depending on the flanking sequences, the C2′?OH group forms hydrogen bonds with the backbone, 3′‐neighboring base, and/or sugar. Interestingly, even in similar purine‐rG‐pyrimidine environments (A‐rG‐C and G‐rG‐C), a riboguanosine unit affects DNA in a distinct manner and manifests different hydrogen bonds, which makes generalizations difficult.     

Sequence,Chromatin and Evolution of Satellite DNA

Satellite DNA consists of abundant tandem repeats that play important roles in cellular processes, including chromosome segregation, genome organization and chromosome end protection. Most satellite DNA repeat units are either of nucleosomal length or 5–10 bp long and occupy centromeric, pericentromeric or telomeric regions. Due to high repetitiveness, satellite DNA sequences have largely been absent from genome assemblies. Although few conserved satellite-specific sequence motifs have been identified, DNA curvature, dyad symmetries and inverted repeats are features of various satellite DNAs in several organisms. Satellite DNA sequences are either embedded in highly compact gene-poor heterochromatin or specialized chromatin that is distinct from euchromatin. Nevertheless, some satellite DNAs are transcribed into non-coding RNAs that may play important roles in satellite DNA function. Intriguingly, satellite DNAs are among the most rapidly evolving genomic elements, such that a large fraction is species-specific in most organisms. Here we describe the different classes of satellite DNA sequences, their satellite-specific chromatin features, and how these features may contribute to satellite DNA biology and evolution. We also discuss how the evolution of functional satellite DNA classes may contribute to speciation in plants and animals.     

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress

DNA can experience “replication stress”, an important source of genome instability, induced by various external or endogenous impediments that slow down or stall DNA synthesis. While genome instability is largely documented to favor both tumor formation and heterogeneity, as well as drug resistance, conversely, excessive instability appears to suppress tumorigenesis and is associated with improved prognosis. These findings support the view that karyotypic diversity, necessary to adapt to selective pressures, may be limited in tumors so as to reduce the risk of excessive instability. This review aims to highlight the contribution of specialized DNA polymerases in limiting extreme genetic instability by allowing DNA replication to occur even in the presence of DNA damage, to either avoid broken forks or favor their repair after collapse. These mechanisms and their key regulators Rad18 and Polθ not only offer diversity and evolutionary advantage by increasing mutagenic events, but also provide cancer cells with a way to escape anti-cancer therapies that target replication forks.     

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn²⁺ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg²⁺. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn²⁺ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn²⁺ activated the reaction a thousand-fold compared to Mg²⁺, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn²⁺ compared to Mg²⁺. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn²⁺ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.     

Ruthenium(II) Complex-Based G-quadruplex DNA Selective Luminescent ‘Light-up’ Probe for RNase H Activity Detection

A bis-heteroleptic ruthenium(II) complex, 1[PF₆]₂ of benzothiazole amide substituted 2,2′-bipyridine ligand ( bmbbipy ) has been synthesized for the selective detection of G-quadruplex (GQ) DNA and luminescence-assay-based RNase H activity monitoring. Compound 1[PF₆]₂ exhibited aggregation-caused quenching (ACQ) in water. Aggregate formation was supported by DLS, UV-vis, and ¹H NMR spectroscopy results, and the morphology of aggregated particles was witnessed by SEM and TEM. 1[PF₆]₂ acted as an efficient GQ DNA-selective luminescent light-up probe over single-stranded and double-stranded DNA. The competency of 1[PF₆]₂ for selective GQ structure detection was established by PL and CD spectroscopy. For 1[PF₆]₂ , the PL light-up is exclusively due to the rigidification of the benzothiazole amide side arm in the presence of GQ-DNA. The interaction between the probe and GQ-DNA was analyzed by molecular docking analysis. The GQ structure detection capability of 1[PF₆]₂ was further applied in the luminescent ‘off-on’ RNase H activity detection. The assay utilized an RNA:DNA hybrid, obtained from 22AG2-RNA and 22AG2-DNA sequences. RNase H solely hydrolyzed the RNA of the RNA:DNA duplex and released G-rich 22AG2-DNA, which was detected via the PL enhancement of 1[PF₆]₂ . The selectivity of RNase H activity detection over various other restriction enzymes was also demonstrated.     

Verbesserte Einsatzmöglichkeiten des 4-Methoxybenzylrestes als 2′-OH-Schutz bei Ribonucleotidsynthesen durch TFA-Acidolyse

An Improved Method for the Application of the 4-Methoxybenzyl Group to Protect the 2′-Hydroxyl Group in the Ribonucleotide Synthesis by TFA-acidolysis The cleavage of the 4-methoxybenzyl group from the 2′-OH-position of ribonucleosides by the hydrogenation with different Pd-catalysts as well as trifluoracetic acid has been studied in detail. During hydrogenation, side reactions at the base residue of cytidine-occurred which, however, could be extensively suppressed by PdCl₂ catalysis. More practicable results were obtained with trifluoracetic acid in the presence of cation scavengers, allowing smoothly to convert a series of 2′-methoxybenzyl ribonucleotides to the homogeneous deprotection products.     

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.     

Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H

To construct a DNA-linked RNase H, which cleaves RNA site-specificallyat high temperatures, the 15-mer DNA, which is complementaryto the polypurine-tract sequence of human immunodeficiency virus-1RNA (PPT-RNA), was cross-linked to the unique thiol group ofCys135 in the Thermus thermophilus RNase HI variant. The resultantDNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH,in which the RNase H portion of the d15-C135/TRNH is replacedby the Escherichia coli RNase HI variant, cleaved the 15-merPPT-RNA site-specifically. The mixture of the unmodified enzymeand the unlinked 15-mer DNA also cleaved the PPT-RNA but ina less strict manner. In addition, this mixture cleaved thePPT-RNA much less effectively than the DNA-linked enzyme. Theseresults indicate that the cross-linking limits but acceleratesthe interaction between the enzyme and the DNA/RNA substrate.The d15-C135/TRNH cleaved the PPT-RNA more effectively thanthe d15-C135/ERNH at temperatures higher than 50°C. Thed15-C135/TRNH showed the highest activity at 65°C, at whichthe d15-C135/ERNH showed little activity. Such a thermostableDNA-linked RNase H may be useful to cleave RNA molecules withhighly ordered structures in a sequence-specific manner.     

Chl1, an ATP-Dependent DNA Helicase,Inhibits DNA:RNA Hybrids Formation at DSB Sites to Maintain Genome Stability in S. pombe

As an ATP-dependent DNA helicase, human ChlR1/DDX11 (Chl1 in yeast) can unwind both DNA:RNA and DNA:DNA substrates in vitro. Studies have demonstrated that ChlR1 plays a vital role in preserving genome stability by participating in DNA repair and sister chromatid cohesion, whereas the ways in which the biochemical features of ChlR1 function in DNA metabolism are not well understood. Here, we illustrate that Chl1 localizes to double-strand DNA break (DSB) sites and restrains DNA:RNA hybrid accumulation at these loci. Mutation of Chl1 strongly impairs DSB repair capacity by homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways, and deleting RNase H further reduces DNA repair efficiency, which indicates that the enzymatic activities of Chl1 are needed in Schizosaccharomyces pombe. In addition, we found that the Rpc37 subunit of RNA polymerase III (RNA Pol III) interacts directly with Chl1 and that deletion of Chl1 has no influence on the localization of Rpc37 at DSB site, implying the role of Rpc37 in the recruitment of Chl1 to this site.     

UV Differentially Induces Oxidative Stress,DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

Chronic myeloid leukemia (CML) cells express the active BCR-ABL1 protein, which has been targeted by imatinib in CML therapy, but resistance to this drug is an emerging problem. BCR-ABL1 induces endogenous oxidative stress promoting genomic instability and imatinib resistance. In the present work, we investigated the extent of oxidative stress, DNA damage, apoptosis and expression of apoptosis-related genes in BCR-ABL1 cells sensitive and resistant to imatinib. The resistance resulted either from the Y253H mutation in the BCR-ABL1 gene or incubation in increasing concentrations of imatinib (AR). UV irradiation at a dose rate of 0.12 J/(m²·s) induced more DNA damage detected by the T4 pyrimidine dimers glycosylase and hOGG1, recognizing oxidative modifications to DNA bases in imatinib-resistant than -sensitive cells. The resistant cells displayed also higher susceptibility to UV-induced apoptosis. These cells had lower native mitochondrial membrane potential than imatinib-sensitive cells, but UV-irradiation reversed that relationship. We observed a significant lowering of the expression of the succinate dehydrogenase (SDHB) gene, encoding a component of the complex II of the mitochondrial respiratory chain, which is involved in apoptosis sensing. Although detailed mechanism of imatinib resistance in AR cells in unknown, we detected the presence of the Y253H mutation in a fraction of these cells. In conclusion, imatinib-resistant cells may display a different extent of genome instability than their imatinib-sensitive counterparts, which may follow their different reactions to both endogenous and exogenous DNA-damaging factors, including DNA repair and apoptosis.     

Kinase Activity of PAR1b,Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor,Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell–cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.     

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

A series of DNA-linked RNases H, in which the 15-mer DNA iscross-linked to the Thermus thermophilus RNase HI (TRNH) variantsat positions 135, 136, 137 and 138, were constructed and analyzedfor their abilities to cleave the complementary 15-mer RNA.Of these, that with the DNA adduct at position 135 most efficientlycleaved the RNA substrate, indicating that position 135 is themost appropriate cross-linking site among those examined. Toexamine whether DNA-linked RNase H also site-specifically cleavesa highly structured natural RNA, DNA-linked TRNHs with a seriesof DNA adducts varying in size at position 135 were constructedand analyzed for their abilities to cleave MS2 RNA. These DNAadducts were designed such that DNA-linked enzymes cleave MS2RNA at a loop around residue 2790. Of the four DNA-linked TRNHswith the 8-, 12-, 16- and 20-mer DNA adducts, only that withthe 16-mer DNA adduct efficiently and site-specifically cleavedMS2 RNA. Primer extension revealed that this DNA-linked TRNHcleaved MS2 RNA within the target sequence.