Overcoming variant mutation-related impacts on viral sequencing and detection methodologies.

Prompt and accurate pathogen identification, by diagnostics and sequencing, is an effective tool for tracking and potentially curbing pathogen spread. Targeted detection and amplification of viral genomes depends on annealing complementary oligonucleotides to genomic DNA or cDNA. However, genomic mutations that occur during viral evolution may perturb annealing, which can result in incomplete sequence coverage of the genome and/or false negative diagnostic test results. Herein, we demonstrate how to assess, test, and optimize sequencing and detection methodologies to attenuate the negative impact of mutations on genome targeting efficiency. This evaluation was conducted using in vitro-transcribed (IVT) RNA as well as RNA extracted from clinical SARS-CoV-2 variant samples, including the heavily mutated Omicron variant. Using SARS-CoV-2 as a current example, these results demonstrate how to maintain reliable targeted pathogen sequencing and how to evaluate detection methodologies as new variants emerge.

A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution.

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

Fidelity index determination of DNA methyltransferases.

DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required.

Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases.

In T4 bacteriophage, 5-hydroxymethylcytosine (5hmC) is incorporated into DNA during replication. In response, bacteria may have developed modification-dependent type IV restriction enzymes to defend the cell from T4-like infection. PvuRts1I was the first identified restriction enzyme to exhibit specificity toward hmC over 5-methylcytosine (5mC) and cytosine. By using PvuRts1I as the original member, we identified and characterized a number of homologous proteins. Most enzymes exhibited similar cutting properties to PvuRts1I, creating a double-stranded cleavage on the 3' side of the modified cytosine. In addition, for efficient cutting, the enzymes require two cytosines 21-22-nt apart and on opposite strands where one cytosine must be modified. Interestingly, the specificity determination unveiled a new layer of complexity where the enzymes not only have specificity for 5-ß-glucosylated hmC (5ßghmC) but also 5-α-glucosylated hmC (5αghmC). In some cases, the enzymes are inhibited by 5ßghmC, whereas in others they are inhibited by 5αghmC. These observations indicate that the position of the sugar ring relative to the base is a determining factor in the substrate specificity of the PvuRts1I homologues. Lastly, we envision that the unique properties of select PvuRts1I homologues will permit their use as an additive or alternative tool to map the hydroxymethylome.

High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells.

We describe the use of a unique DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5-hydroxymethylcytosine (5hmC) at low-occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follow the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the monomethylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

Substrate recognition by ß-ketoacyl-ACP synthases.

ß-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ß-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways.

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.