S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.

R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

Mph1 and Mus81-Mms4 prevent aberrant processing of mitotic recombination intermediates.

Homology-dependent repair of double-strand breaks (DSBs) from nonsister templates has the potential to generate loss of heterozygosity or genome rearrangements. Here we show that the Saccharomyces cerevisiae Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage. A role for Yen1 is only apparent in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair of a single DSB, suggesting that the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules (JMs) accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the mus81Δ mutant contain a single Holliday junction (HJ) explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize an early strand exchange intermediate and direct repair to noncrossover or crossover outcomes, respectively.

A label-free protamine-assisted colorimetric sensor for highly sensitive detection of S1 nuclease activity.

A label-free, sensitive, simple and general colorimetric method was reported to monitor S1 nuclease activity based on protamine-assisted aggregation of gold nanoparticles (AuNPs). Here, protamine, a linear polycation, was used as a medium for causing the aggregation of negatively charged AuNPs by electrostatic interactions, resulting in changes in the surface plasmon resonance (SPR) absorption bands as well as the color of AuNPs. Here, the AuNPs were employed as an indicator to detect the level of S1 nuclease in the solution. Substrate DNA could be cleaved into small fragments by the specific S1 nuclease, which effectively prevents the electrostatic interaction between DNA and protamine and thus facilitates the interaction between protamine and AuNPs. The quantitative analysis of S1 nuclease activity can be performed via directly measuring the changes in the absorption spectra of the AuNPs. Using S1 nuclease as a model analyte, the limit of detection was estimated to be 1.0 × 10-4 U mL-1. Furthermore, the proposed concept has been successfully applied in S1 nuclease analysis of serum samples, offering an ultrasensitive strategy for the speedy detection of the nuclease activity and providing a new avenue for high-throughput screening of nucleases and drugs with potential inhibition properties.

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Heterologous expression and characterization of Penicillium citrinum nuclease P1 in Aspergillus niger and its application in the production of nucleotides.

Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg2+, Zn2+ and Cu2+, and inhibited by addition of Ca2+, Fe2+, Mn2+, Ni2+, Co2+, Mg2+, K+ and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5'- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.

Endonucleases responsible for DNA repair of helix-distorting DNA lesions in the thermophilic crenarchaeon Sulfolobus acidocaldarius in vivo.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

The effects of DNA supercoiling on G-quadruplex formation.

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.

Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast.

The Saccharomyces cerevisiae Rad1/Rad10 complex is a multifunctional, structure-specific endonuclease that processes UV-induced DNA lesions, recombination intermediates, and inter-strand DNA crosslinks. However, we do not know how Rad1/Rad10 recognizes these structurally distinct target molecules or how it is incorporated into the protein complexes capable of incising divergent substrates. Here, we have determined the order and hierarchy of assembly of the Rad1/Rad10 complex, Saw1, Slx4, and Msh2/Msh3 complex at a 3' tailed recombination intermediate. We found that Saw1 is a structure-specific DNA binding protein with high affinity for splayed arm and 3'-flap DNAs. By physical interaction, Saw1 facilitates targeting of Rad1 at 3' tailed substrates in vivo and in vitro, and enhances 3' tail cleavage by Rad1/Rad10 in a purified system in vitro. Our results allow us to formulate a model of Rad1/Rad10/Saw1 nuclease complex assembly and 3' tail removal in recombination.

Differential Enzymatic 16O/18O Labeling for the Detection of Cross-Linked Nucleic Acid-Protein Heteroconjugates.

Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential 16O/18O-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry.

Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae.

Methylation of ribose sugars at the 2'-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2'-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5' central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D' box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

Saw1 localizes to repair sites but is not required for recruitment of Rad10 to repair intermediates bearing short non-homologous 3' flaps during single-strand annealing in S. cerevisiae.

SAW1 is required for efficient removal by the Rad1-Rad10 nuclease of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break (DSB) repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing. Saw1 was shown in vitro to bind flaps with high affinity, but displayed diminished affinity when flaps were short (<30 deoxynucleotides [nt]), consistent with it not being required for short flap cleavage. Accordingly, this study, using in vivo fluorescence microscopy showed that SAW1 was not required for recruitment of Rad10-YFP to DNA DSBs during their repair by SSA when the flaps were ~10 nt. In contrast, recruitment of Rad10-YFP to DSBs when flaps were ~500 nt did require SAW1 in G1 phase of cell cycle. Interestingly, we observed a substantial increase in colocalization of Saw1-CFP and Rad10-YFP at DSBs when short flaps were formed during repair, especially in G1, indicating significant recruitment of Saw1 despite there being no requirement for Saw1 to recruit Rad10. Saw1-CFP was seldom observed at DSBs without Rad10-YFP. Together, these results support a model in which Saw1 and Rad1-Rad10 are recruited as a complex to short and long flaps in all phases of cell cycle, but that Saw1 is only required for recruitment of Rad1-Rad10 to DSBs when long flaps are formed in G1.

Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates.

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.

Development of enzyme technology for Aspergillus oryzae, A. sojae, and A. luchuensis, the national microorganisms of Japan.

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T1 (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S1 (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man10GlcNAc2 and Man11GlcNAc2 were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.

Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages.

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.

Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters.

S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uµl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uµl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis.

Enhancement of nuclease P1 production by Penicillium citrinum YL104 immobilized on activated carbon filter sponge.

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

HsOrc4-Dependent Dna Remodeling of the ori-ß Dhfr Replicator.

Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-ß sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-ß DHFR sequence. We show that the topologically closed DHFR ori-ß replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases.