Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.

Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases.

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

Rapid Time Scale Analysis of T4 DNA Ligase-DNA Binding.

DNA ligases, essential to both in vivo genome integrity and in vitro molecular biology, catalyze phosphodiester bond formation between adjacent 3'-OH and 5'-phosphorylated termini in dsDNA. This reaction requires enzyme self-adenylylation, using ATP or NAD+ as a cofactor, and AMP release concomitant with phosphodiester bond formation. In this study, we present the first fast time scale binding kinetics of T4 DNA ligase to both nicked substrate DNA (nDNA) and product-equivalent non-nicked dsDNA, as well as binding and release kinetics of AMP. The described assays utilized a fluorescein-dT labeled DNA substrate as a reporter for ligase·DNA interactions via stopped-flow fluorescence spectroscopy. The analysis revealed that binding to nDNA by the active adenylylated ligase occurs in two steps, an initial rapid association equilibrium followed by a transition to a second bound state prior to catalysis. Furthermore, the ligase binds and dissociates from nicked and nonsubstrate dsDNA rapidly with initial association affinities on the order of 100 nM regardless of enzyme adenylylation state. DNA binding occurs through a two-step mechanism in all cases, confirming prior proposals of transient binding followed by a transition to a productive ligase·nDNA (Lig·nDNA) conformation but suggesting that weaker nonproductive "closed" complexes are formed as well. These observations demonstrate the mechanistic underpinnings of competitive inhibition by rapid binding of nonsubstrate DNA, and of substrate inhibition by blocking of the self-adenylylation reaction through nick binding by deadenylylated ligase. Our analysis further reveals that product release is not the rate-determining step in turnover.

Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

A network of PUF proteins and Ras signaling promote mRNA repression and oogenesis in C. elegans.

Cell differentiation requires integration of gene expression controls with dynamic changes in cell morphology, function, and control. Post-transcriptional mRNA regulation and signaling systems are important to this process but their mechanisms and connections are unclear. During C. elegans oogenesis, we find that two groups of PUF RNA binding proteins (RNABPs), PUF-3/11 and PUF-5/6/7, control different specific aspects of oocyte formation. PUF-3/11 limits oocyte growth, while PUF-5/6/7 promotes oocyte organization and formation. These two PUF groups repress mRNA translation through overlapping but distinct sets of 3' untranslated regions (3'UTRs). Several PUF-dependent mRNAs encode other mRNA regulators suggesting both PUF groups control developmental patterning of mRNA regulation circuits. Furthermore, we find that the Ras-MapKinase/ERK pathway functions with PUF-5/6/7 to repress specific mRNAs and control oocyte organization and growth. These results suggest that diversification of PUF proteins and their integration with Ras-MAPK signaling modulates oocyte differentiation. Together with other studies, these findings suggest positive and negative interactions between the Ras-MAPK system and PUF RNA-binding proteins likely occur at multiple levels. Changes in these interactions over time can influence spatiotemporal patterning of tissue development.

Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA ligase.

T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5'-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5'-phosphate or a 5'-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5'-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4±0.1 s(-1) and 150±50 µm(-1) s(-1), respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded kcat and kcat/Km values of 0.64±0.08 s(-1) and 240±40 µm(-1) s(-1). Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s(-1), respectively, with the slowest step ∼10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s(-1), also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (∼6 s(-1)), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion.

A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase.

Mismatch repair in Escherichia coli involves a number of proteins including MutL and UvrD. Eukaryotes also possess MutL homologues; however, no UvrD helicase homologues have been identified. The hyperthermophilic bacterium Aquifex aeolicus has a MutL protein (Aae MutL) that possesses a latent endonuclease activity similar to eukaryotic, but different from E. coli, MutL proteins. By sequence homology Aq793 is a member of the PcrA/UvrD/Rep helicase subfamily. We expressed Aae MutL and the putative A. aeolicus DNA helicase (Aq793) proteins in E. coli. Using synthetic oligonucleotide substrates, we observed that lower concentrations of Aq793 were required to unwind double-stranded DNA that had a 3'-poly(dT) overhang as compared with double-stranded DNA with a 5'-poly(dT) or lacking a poly(dT) tail. This unwinding activity was stimulated by adding Aae MutL with maximal stimulation observed at an approximately 1.5:1 (MutL:Aq793) stoichiometric ratio. The enhancement of Aq793 helicase activity did not require the Aae MutL protein to retain endonuclease activity. Furthermore, the C-terminal 123 amino acid residues of Aae MutL were sufficient to stimulate Aq793 helicase activity, albeit at a significantly reduced efficacy. To the best of our knowledge this is the first time a human PMS2 homologue has been demonstrated to stimulate a PcrA/UvrD/Rep subfamily helicase, and this finding may further our understanding of the evolution of the mismatch repair pathway.

Corrigendum: Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.

[This corrects the article DOI: 10.3389/fmolb.2021.778400.].

Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes. Within eukaryotes, a wide array of evolutionary diversity exists. Humans possess all four Y-family pols (pols η, ι, κ, and Rev1), Schizosaccharomyces pombe has three Y-family pols (pols η, κ, and Rev1), and Saccharomyces cerevisiae only has polη and Rev1. Here, we report the cloning, expression, and biochemical characterization of the four Y-family pols from the lower eukaryotic thermophilic fungi, Thermomyces lanuginosus. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human polÉ©) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that polι originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes.

Protein splicing elements and plants: from transgene containment to protein purification.

Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they mediate the protein splicing process. Because inteins are natural protein engineering elements they have been harnessed for use in a number of applications, including protein purification, protein semisynthesis, and in vivo and in vitro protein modifications. This review focuses on the use of inteins in plants. A split-gene technique utilizes inteins to reconstitute the activity of a transgene product with the goal of limiting the spread of transgenes from a genetically modified plant to a weedy relative. Furthermore, merging the intein tag for protein purification with the large protein yields possible with plants has the potential to produce pharmaceutically important proteins. Finally, relevant techniques that may be used in plants in the future are discussed.

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.

DNA damage in preserved specimens and tissue samples: a molecular assessment.

The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage.Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents - such as light, oxygen or formaldehyde - on free nucleotides.We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS.We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.

Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro.

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.

Response to Comment on "DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification".

Following the Comment of Stewart et al, we repeated our analysis on sequencing runs from The Cancer Genome Atlas (TCGA) using their suggested parameters. We found signs of oxidative damage in all sequence contexts and irrespective of the sequencing date, reaffirming that DNA damage affects mutation-calling pipelines in their ability to accurately identify somatic variations.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis.

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.