RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1? rnh201? cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1? rnh201? cells, but RNase H is not generally required for efficient DSB repair.     

Effect of Ischemia on the Activity of DNA-Dependent RNA Polymerase and DNA Polymerase

Abstract: Ischemia, anoxia, and hypoxia of the brain have been shown to inhibit protein synthesis in the central nervous system. To obtain data on the changes in DNA-dependent RNA and DNA polymerases as they pertain specifically to neurons and glia, nuclear enriched neuronal and glial fractions were prepared, by sucrose-gradient centrifugation, from spinal cords of adult dogs that had been subjected to prolonged ischemia. The isolated fractions were assayed for enzyme activity by a radiochemical technique. RNA polymerase was affected more than DNA polymerase, activity being reduced considerably in both neurons and glia. Possible causes of the difference in sensitivity to ischemia are discussed.     

一种简便快速的聚合酶活性实时检测新方法

基于双链DNA结合染料能特异嵌入双链DNA发出荧光的原理,发展了一种实时检测DNA聚合酶活性的简便方法.在检测过程中,聚合酶的聚合反应进程被实时转换为荧光信号,通过监测荧光强度的变化实时检测聚合酶的活性及药物对聚合酶活性的影响.该方法不需要对DNA进行放射性同位素标记和荧光标记,也不需要聚丙烯酰胺凝胶电泳和聚合酶链式反应,是一种简便、快速的聚合酶活性实时检测新方法,为研究抗肿瘤药物对聚合酶活性的影响提供了一种简捷方法,也将为相关疾病诊治和药物筛选提供一种新的思路.     

Colorimetric Assay for Uracil DNA Glycosylase Activity Based on Toehold‐Mediated Strand Displacement Circuit

Herein, a novel enzyme‐free and label‐free strategy for colorimetric assay of uracil DNA glycosylase (UDG) activity, which relies on a target‐activated toehold‐mediated strand displacement (TMSD) circuit is described. The strategy employs a detection duplex probe composed of a uracil‐containing strand (US) and a catalyst strand (CS). UDG present in a sample will cleave uracil bases within US and destabilize the detection duplex probe, which then leads to the dissociation of the detection duplex, releasing CS. The free CS promotes the TMSD reaction, consequently liberating a G‐quadruplex DNAzyme strand (GS) which is initially caged by a blocker strand (BS). Notably, a fuel strand (FS) is supplemented to recycle the CS to promote another cycle of TMSD reaction. As a consequence, a large number of GSs are activated by UDG activity and a distinct colorimetric signal is produced through the oxidation of ABTS promoted by the peroxidase mimicking activity of the liberated GSs. Based on this design principle, UDG activity down to 0.006 U mL^?1 with excellent selectivity is successfully determined. The practical applicability of this assay is also demonstrated by reliably determining UDG activities in human serum.     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.     

Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1

Metagenome‐derived LC11‐RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto‐RNase H1). It lacks a C‐terminal tail, which is responsible for hyperstabilization of Sto‐RNase H1. Sto‐RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double‐stranded RNA (dsRNA). To examine whether LC11‐RNase H1 also exhibits both RNase H and dsRNase activities, LC11‐RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11‐RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto‐RNase H1. However, LC11‐RNase H1 did not exhibit dsRNase activity at any condition examined. LC11‐RNase H1 was less stable than Sto‐RNases H1 and its derivative lacking the C‐terminal tail (Sto‐RNase H1ΔC6) by 37 and 13°C in T_m, respectively. To understand the structural bases for these differences, the crystal structure of LC11‐RNase H1 was determined at 1.4 Å resolution. The LC11‐RNase H1 structure is highly similar to the Sto‐RNase H1 structure. However, LC11‐RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA‐phosphate binding pocket, while Sto‐RNase H1 has one groove containing the active site. In addition, LC11‐RNase H1 contains more cavities and buried charged residues than Sto‐RNase H1. We propose that LC11‐RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11‐RNase H1 is less stable than Sto‐RNase H1ΔC6 because of the increase in cavity volume and number of buried charged residues.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

Sequence-constructive SELEX: A new strategy for screening DNA aptamer binding to Globo H

We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5′ and 3′-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures.     

一种利用Taq酶快速标记DNA探针的方法

目的：探索低成本、高效、快速的DNA探针标记方法。方法：以特定引物和16nler的随机引物作为延伸引物,利用Taq酶标记DNA探针。以大肠杆菌Klenow片断随机引物延伸标记法作为对照。点杂交方法检测探针标记效果。结果与结论：Taq酶标记法和大肠杆菌Klenow片段随机引物延伸标记法同样有较好的标记效果,且随机引物或特定引物作为延伸引物均可以合成足够有效的探针。Taq酶标记法是一种低成本、高效、快速的DNA探针标记方法。