Compositions and methods comprising brig2 DNA glycosylase

Abstract

Provided are compositions and methods for improved detection and location of 5- hydroxymethylcytosine (5hmC) modifications of DNA. A protein used in the method is a DNA glycosylase provided is referred to as "Brig2." Catalytically inactive versions of Brig2 are also provided. The Brig2 proteins can be used in a variety of methods that involve isolation of DNA comprising 5hmC modifications, and determining the location of the 5hmC by incorporating use of the Brig2 proteins.

Description

[0001]Attorney Docket 076091.00180 COMPOSITIONS AND METHODS COMPRISING BRIG2 DNA GLYCOSYLASE CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. provisional application no.63 / 634,586, filed April 16, 2024, the entire disclosure of which is incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2025, is named “076091_00180_ST26.xml”, and is 5,328 bytes in size. RELATED INFORMATION Double stranded DNA, such as genomic DNA, often includes 5-hydroxymetnyl- cytosine (“5hmC”) nucleobases. Such modifications are common in the human genome, especially within neurons, as well as other cell types. While there are existing methods for detecting and locating 5hmC modifications, they are cumbersome and require numerous reagents and steps. There is thus a need for improved compositions and methods for analysis of 5hmC modifications, the disclosure is pertinent to this need. BRIEF SUMMARY The disclosure provides compositions and methods for improved detection and location of 5hmC modifications. In an example, the disclosure provides a DNA glycosylase, and catalytically inactive derivatives thereof. The DNA glycosylase is referred to herein as “Brig2. “ The catalytically inactive derivative is referred to as “dBrig2.” The described Brig2 proteins can be used in a variety of methods that involve isolation of DNA comprising 5hmC modifications, and determining the location of the 5hmC modifications by adapting and modifying existing sequencing reagents and methods by incorporating use of the Brig2 proteins. In examples, the disclosure thus provides an isolated or recombinantly produced Brig2 protein that comprises or consists of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1. In examples, the isolated or recombinantly produced Brig2 protein comprises a change in amino acid position in SEQ ID NO:1 at position 127 (an aforementioned dBrig2 protein), wherein the change is to an amino acid other than D, and wherein the Brig2 protein comprising the amino acid change has reduced or eliminated capability to excise 5-hydroxymethylcytosine (5hmC) nucleobases to generate abasic sites. The Brig2 protein can be modified to include additional amino acids, which may comprise a purification tag. In examples, a described Brig2 protein may be modified such that it comprise a detectable label, and / or or the Brig2 protein is present in a fusion protein. In examples, a described Brig2 protein may be attached to a solid substrate and / or is may be present in a complex with a 5-hydroxymethylcytosine nucleobase that is present on a DNA polynucleotide. In examples, the solid substrate may be made of any of glass, plastic, gold, or polymers. The described substrate may be covalently or non-covalently attached to the substrate. In examples, the solid substrate may be provided in the form of nanoparticles or beads. In examples, the solid substrate may be present in a column. In examples the disclosure provides a method comprising contacting a DNA sample with a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 to generate abasic sites at 5hmC nucleobases with the DNA sample. Isolated DNA samples that comprise the generated abasic sites are included in the disclosure. The disclosure provides for determining the location of the generated abasic sites in the DNA sample. In an example, the DNA sample is contacted with a dBrig2 protein to bind to 5-hydroxymethylcytosine nucleobases, prior to determining the location of abasic sites in the DNA sample. In examples, the disclosure provides a kit comprising a sealed container and a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1. The kit may include one or more sealed containers that contain reagents for use of the protein for determining the presence and / or location of 5-hydroxymethylcytosine nucleobases in a DNA sample. In examples, the disclosure provides an isolated polynucleotide encoding a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1. In examples, the isolated polynucleotide may be present in any suitable expression vector, including but not necessarily limited to a plasmid which may have been engineered to encode the protein. In an example, the disclosure provides a method comprising allowing expression of a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 from an expression vector introduced into a population of cells in vitro. The cells may be any suitable cells for use in protein production, and may be prokaryotic cells, or eukaryotic cells. In examples, the population of cells comprises bacteria that have been engineered to produce the described protein. The disclosure provides separating the Brig2 protein from the population of cells. The protein may be purified to any desirable degree of purity. The disclosure includes a purified Brig2 protein preparation that has been isolated from the cells that have bene configured to produce the protein. BRIEF DESCRIPTION OF THE FIGURES The disclosure includes all aspects of the disclosure as depicted on the accompanying figures. FIG 1. Screening of an environmental DNA (eDNA) library uncovers an unknown gene that protects E. coli against phage T4 Δagt Δbgt, a T4 mutant phage thatlacks glucosylation of 5hmC nucleobases. (A-D) Tenfold serial dilutions of different phageson lawns of E. coli EC100 that harbor pWEB-TNC or pAM38 carrying different genes present in the defense fragment. (A) pWEB-TNC carrying full resistant clone A or fragments of clone A challenged against T4 Δagt Δbgt, T4 or T4 (C) (a mutant T4 that carries cytosine instead of 5hmC). Specific genes expressed by each cosmid are depicted to the right. (B) Similar to (A) but phages used for infection are T2, T6, and T6 escaper-1 (a mutant T6 phage with non-glucosylated 5hmC). (C) Vector pAM38 overexpressing the identified defense gene, encoding Brig2, upon arabinose induction. (D) pWEB-TNC carrying the gene encoding Brig2 or the genes encoding Brig2 and BapA, and with stop codons genetically inserted into each gene as indicated (XX). FIG 2. Brig2 provides defense against phages with 5-hydroxymethylcytosine DNA modifications. (A-C) Tenfold serial dilutions of different phages on lawns of E. coli EC100 that harbor the cosmid pWEB-TNC expressing Brig2 (pBrig2) or both Brig2 andBapA (pBrig2-BapA). (A) Infection with T-even phages in the BASEL collection. (B) E. coliharboring pWEB-TNC, pBrig2, and pBrig2-BapA and complemented with plasmids carrying phage T4 glucosyltranferase genes (alpha or beta) or an empty vector and challenged againstphage T4 Δagt Δbgt or T4. (C) E. coli harboring pWEB-TNC and pBrig2 were complemented with a plasmid carrying the T4 beta-glucosyltranferase gene or an empty vector and challenged against phage T4 Δbgt. FIG.3: Brig2 targets 5hmC in phage DNA. (A) Agarose gel electrophoresis of 60- nucleotide (nt) single-stranded oligonucleotides containing a single modified base, incubated with either hSMUG1 or Brig2. (B) Same as (A) but heated with NaOH after the initial incubation step. dU, deoxyuridine; dC, deoxycytidine; 5-mC, 5-methylcytosine; 5-hmC, 5- hydroxymethylcytosine. (C) Polyacrylamide gel electrophoresis of increasing concentrations of Brig2 incubated with a 60-nt nucleotide oligo containing 5hmC. (D) Same as (C) but heated with NaOH after initial incubation step. (E) Polyacrylamide gel electrophoresis of increasing concentrations of Brig2 incubated with a 60-nt nucleotide oligo containing 5hmC or dU. Gel shows the reactions after being heated with NaOH. (F) Graph showing the variation of the upper band intensity for dU and 5hmC oligonucleotides from the gel in (E) plotted against Brig2 concentration. FIG.4: Brig2 degrades phage DNA containing 5hmC nucleobases. (A-D) Agarose gel electrophoresis of phage DNA incubated with increasing concentrations of Brig2 at 37 ^C for 30 minutes. (A-B) T4, T4 Δagt Δbgt and control pWEB-TNC DNA incubated with Brig2. Top gel (A) shows DNA that was incubated with the enzyme and bottom gel (B) shows DNA that was incubated with the enzyme and then heated at 65 ^C for 20 minutes after initial incubation step. (C-D) T4Δagt and T4 Δbgt phage DNA incubated with Brig2. Top gel (C) shows DNA that was incubated with the enzyme and bottom gel (D) shows DNA that was incubated with the enzyme and then heated at 65 ^C for 20 minutes after initial incubation step. FIG.5. Selection of soil DNA library fragments that provide immunity against mutant T4 phage with non-glucosylated 5hmC nucleobases. (A) Tenfold serial dilutions of phages T5 and T4 Δagt Δbgt on lawns of E. coli EC100 colonies with different soil DNA libraries clones, following selection with T4 Δagt Δbgt. (B) Genetic map of the full metagenomic eDNA fragment in the identified resistant clone A that contains Brig2. Prediction of the encoded genes and fragments used in cloning for narrowing down the defense gene of interest (Brig2) are annotated. (C) Tenfold serial dilutions of phage T4 Δagt Δbgt on lawns of E. coli EC100 colonies with pWEB-TNC carrying the full resistant clone A or fragments of this clone. FIG.6. Brig2 immunity against wild-type and mutant T4 phages via liquidculture assays. (A-F) Growth curves of E. coli EC100 strains carrying pWEB-TNC withBrig2 and BapA (pBrig2-BapA), or the same construct with genetically encoded stop codons in the indicated gene, depicted as STOP. (A) Strains grown with no phage. (B-C) Strains infected with phage T4 Δagt Δbgt at MOI 1 (B) and MOI 10 (C). (D-F) Strains infected with phage T4 at MOI 0.01 (D), MOI 1 (E) and MOI 10 (F). FIG. 7. Brig2 immunity against T2 and T6 phages via liquid culture assays. (A-F) Growth curves of E. coli EC100 strains carrying pWEB-TNC with Brig2 and BapA (pBrig2-BapA), or the same construct with genetically encoded stop codons in the indicated gene, depicted as STOP. (A) Strains grown with no phage. (B-C) Strains infected with phage T2 at MOI 1 (B) and MOI 10 (C). (D-F) Strains infected with phage T6 at MOI 0.01 (D), MOI 1 (E), and MOI 10 (F). FIG. 8. Brig2 predicted structure and active site. (A) AlphaFold2 structural modelof Brig2, colored by pLDDT (a per-residue model confidence score). Red to blue spectrum represents high to low confidence of secondary structure prediction. (B) AlphaFold2 structure of Brig2, colored by N- to C-terminal (blue to red), and predicted active site showing amino acid residues that could be essential for activity. (C) AlphaFold2 structure of the homolog Brig2, colored by N- to C-terminal (blue to red), and the predicted Brig2 active site, showing the residues required for base excision activity. (D) Tenfold serial dilutions of phage T4 Δagt Δbgt on lawns of E. coli EC100 with pWEB-TNC carrying wild-type Brig2 or Brig2 mutants with substitutions in the amino acid residues Y97 and D127 predicted to participate in base excision activity. FIG.9. Activity of Brig2 and the described mutants on oligonucleotides containing 5hmc. WT – wildtype Brig2. D127A / D127A / Y197F = Brig2 mutants / d5hmc – 60 nt oligo with 5hmC. dC – 60 not oligo control with n DNA modifications. L – Ladder (top band 60 nt / mid band 40 nt / lower band 20 nt). FIG.10. Results and analysis of 5hmc sequencing of T2 phage genome using TLS- AP seq. (A) Graphic depicting the process of library preparation for 5hmC sequencing with TLS-AP Seq. The top panel shows four steps of library preparation. Step 3 is an extra step added to NEB Next Ultra II FS Workflow where the process required for 5hmC detection occurs. The expanded inset shows the PCR cycle, which is a single-tube reaction, wherein Brig2 and the polymerase mix are added to generate abasic sites in DNA and repair them. (B) Statistics analysis for a sequencing run. The number of mutations on C or B bases were calculated by comparison of the T2 reference genome. (C) Comparison between the number of steps required to sequence 5hmc with single-nucleotide resolution using a commercially available kit (NEB) and TLS-AP seq. DETAILED DESCRIPTION Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges and other values may be expressed herein as from “about” or “approximately” one particular value, and / or to “about” or “approximately” another particular value. When values are expressed as approximations by the use of the antecedent “about” or “approximately” it will be understood that the particular value forms another example. The term “about” and “approximately” in relation to a numerical value encompasses variations of + / -10%, to + / - 1%. The disclosure includes all steps and reagents such as proteins and nucleic acids, and all combinations of steps reagents, described herein, and as depicted on the accompanying figures. The described steps may be performed as described, including but not necessarily sequentially. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included, including but not limited to sequences included by way of sequence alignments. Sequences of from 80.00%-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included. The disclosure includes any protein having at least 80% amino acid sequence identity with a specific amino acid sequence defined herein by way of a sequence identifier or database entry. Percent amino acid sequence identity with respect proteins means the percentage of amino acid residues in another sequence that are identical with the amino acid residues in the defined sequence, after aligning the sequences in the same reading frame and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and optionally not considering any conservative substitutions as part of the sequence identity. The disclosure includes all polynucleotide and all amino acid sequences that are identified herein by way of a database entry. Such sequences are incorporated herein as they exist in the database on the filing date of this application or patent. Amino-acid residue sequences described herein are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus, unless stated differently. Additionally, a dash at the beginning or end of an amino acid sequence may indicate a peptide bond to a further sequence comprising one or more amino-acid residues. The disclosure includes all amino acid sequences that are defined by sequence identifier, but with one or more changed amino acids, relative to a native amino acid sequence. Amino acid changes include conservative changes, such as by changing an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping, and non-conservative changes, such as such as changing an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping. In example the disclosure provides an isolated or recombinantly produced Brig2 protein, and derivatives thereof wherein the derivatives have at least one amino acid change relative to SEQ ID NO:1. Brig2 amino acid sequence: MDALIKESWTLYERHRAAGFVVAPSIPILFFGDSRAYFESKVKVITVGLNPSRVEFPDG DRYLRFAKARDVYPRIMAGDFYQEYLEALDGYFSNHPYGAWFNSFEPMLKGLGASY YKGAPNTALHTDLCSPLATDPTWSRLSREQMERLRPEGTALWHRLVETLMPDLIIVSV AEGHLDNIHFARAGVWETVHTVERENPYRVKLVGLKLGSGKKTALVFGRAANTPFG TVSNADKRRIGESLKGHVYGQ (SEQ ID NO:1) In SEQ ID NO:1, the D at position 127 may be changed to a different amino acid, non-limiting examples of A, G, V, or N, to provide a catalytically dead Brig2, referred to as dBrig2. In examples, the disclosure provides SEQ ID NO:1 wherein position in 127 is other than D results in a Brig2 protein that can bind to a 5hmC moiety, but does not create an abasic site. Thus, a described Brig2 derivative can be used for different purposes relative to the wild type Brig2 that comprises SEQ ID NO:1. In non-limiting examples, the Brig2 derivative can be configured to enrich DNA samples that contain 5hmC moiety by functioning in a manner similar to antibodies that bind to 5hmC. Thus, a Brig2 derivative may be adapted to be attached to a substrate, such as beads, or a column, or other materials used for separation and / or enrichment of DNA polynucleotides that contain 5hmC. In an example, use of a Brig2 derivative binds to a 5hmC but its binding does not create an abasic site, which can be determined by those skilled in the art, such as the DNA treated with a Brig2 derivative not being cleaved with agents such as NaOH and heat. A Brig2 derivative may also be detectably labeled to facilitate, for example, visualization of 5hmC sites that are bound by detectably labeled Brig2. Suitable detectable labels will be known by those skilled in the art, and include, for example, radiolabels. In examples, the Brig2 derivative may be provided as a component of a fusion protein, wherein at least one other component of the fusion protein comprises a detectable protein, examples of which include green fluorescent protein (GFP), fluorescent protein (RFP), blue fluorescent protein (BFP), mCherry, and the like. Combinations of Brig2 proteins comprising Brig2 proteins modified with different detectable labels and / or different detectable proteins are included. Within a fusion protein comprising a Brig2 segment and a detectable protein segment, the segments may be separated by a suitable linking amino acid sequence. In examples, a described Brig2 protein comprises additional amino acids that are added to a described Brig2 protein. In examples, additional amino acids include any one or a combination of a protein purification tag, such as a Sumo or histidine tag, ribosomal skipping sequences, protease recognition sequences, and linker sequences. In an example, a described Brig2 protein is modified to comprise a purification tag that comprises a poly-histidine tag. In examples, 4, 5, 6, or more histidine amino acids can be appended to the N- or C-terminus of the described Brig2 protein. In an example, a His6 tag is used. In examples a described protein is present in a fusion protein with a segment comprising a Cas protein, including but not necessarily limited to Cas9 or dCas9. Complexes comprising the described protein(s) and DNA are provided. In an example, the disclosure provides a polynucleotide encoding wild type Brig2 that comprises or consists of the sequence: gtggatgcgctgatcaaagagtcttggacgttgtatgagaggcatagggctgccggcttcgtcgtggcgccctccatacccatcctattt ttcggcgacagtagggcctacttcgagtcaaaagttaaagtcatcacggtgggcctgaacccttcccgcgtagagttccccgacggtg acaggtatctgaggttcgctaaagctcgagatgtttacccgcggataatggccggggacttctaccaggaatacctcgaagcgcttgac gggtacttttcgaatcacccttacggcgcatggtttaactccttcgaacccatgctgaaggggctcggggccagctattacaaaggggc gccgaacaccgcgctgcacacggacctgtgctcgccgctggcgaccgacccgacgtggagtagactctcgcgagaacagatggaa agactccggcctgagggcacggcgctctggcaccgactggtcgagacgctaatgccggacttgatcatcgtttccgtggccgaaggc cacttggacaacatacacttcgcccgcgcgggagtgtgggagacggttcacactgtcgagcgtgagaacccctaccgcgtgaaactc gtcggcctcaaattaggctcgggcaagaagaccgccctcgtattcggcagggccgccaacacgcccttcggcacggtctcgaacgc tgacaagcggaggataggggagagtttgaaagggcatgtttatggacagtga (SEQ ID NO:2) In examples, a described Brig2 protein of this disclosure is introduced into one or more prokaryotic or eukaryotic cells. In examples, the prokaryotic cells comprise or consist of gram positive, or gram-negative bacteria. The bacteria may be non-pathogenic, or pathogenic. In examples, a described protein is introduced into prokaryotic cells (e.g., bacterial or archaeal cells) in the context of a host, e.g., a human, animal, or plant host, e.g., or the bacteria are a component of a host’s microbiome or are an abnormal component of a microbiome, e.g., a pathogenic bacteria. In some examples, delivery of a protein described herein results in killing of the recipient cell. The protein may kill some or all of the cells, or render the cells non-pathogenic and / or sensitive to one or more antibiotics. In examples, the protein is used as a component of a food or beverage product, including but not limited to fermented food and beverages, and dairy products. In examples, selective delivery to a specific type of bacteria is used by way of a bacteriophage or packaged phagemids that can express a described Brig protein, but wherein the bacteriophage exhibits a specific tropism for a particular type of bacteria. In examples, a Brig2 protein of this disclosure is administered to an individual in a therapeutically effective amount. In examples, a therapeutically effective amount of a composition of this disclosure is used. The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure. In examples, the disclosure is considered suitable for use in any eukaryotic or prokaryotic cells. For use with eukaryotic cells, a described protein may include a nuclear localization signal (NSL). In examples, a nuclear localization signal sequence comprises a nucleoplasm NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO:3) or SV40 NLS having the sequence PKKKRKV (SEQ ID NO:4). Other NLS signals can be used and, in general, for eukaryotic purposes, a nuclear localization signal comprises one or more short sequences of positively charged lysines or arginines. In examples, a described protein or combination is introduced into cells using any suitable approach. In certain aspects the disclosure includes a pharmaceutical formulation comprising a Brig2 protein described herein. A pharmaceutical formulation comprises one or more pharmaceutically acceptable additives, many of which are known in the art. In some examples, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for administration to humans, or to non-human individuals or cells. In an example, results from using a described sequencing approach are illustrated by FIG.9. FIG.9 shows the activity of Brig2 and descripted mutants on oligonucleotides containing 5hmc. In the method used to produce the results, Brig2 wild type and the Brig2 mutants shown under d5hmC and dC were incubated at 37 degrees Ce for 30 minutes with the respective oligonucleotides. NaOH was added to the samples before heating at 90 degrees C for 30 minutes to induce DNA cleavage at the abasic sits. Samples were cleaned with an oligo concentrator and run on a 10% TBE gel. The results show how wildtype Brig2 creates an abasic site on the 5hmC oligo, which is cleaved after NaOH and heat treatment. The Brig2 mutants D127A and D127N do not create an abasic site on the 5hmC oligo, and there is accordingly no DNA cleavage. Binding of D127 to the 5hmC base has been confirmed through protein crystallography with the substrate. In examples, the disclosure provides for determining the presence and / or location of an abasic site, thereby determining the presence and / or the location of 5hmC in the sample before being contacted with Big2. In examples, the disclosure provides for use of described Brig2 and / or dBrig2 for use with 5hmC sequencing, such uses including but not necessarily limited to the following. ILLUMINA Next Generation Sequencing (NGS):1. snAP-seq:In this example, genomic DNA is treated with Brig2 to generate abasic sites at 5hmC nucleobases in a DNA sample. As a control, genomic (or another DNA sample) without Brig2 treatment is used. The DNA is incubated with a Biotin-PEG3-azide probe in the presence of CuBr. This probe reacts with the aldehyde in the abasic site through a hydrazino- iso-Pictet–Spengler (HIPS) reaction, allowing for tagging and enrichment of abasic sites. A first adapter is ligated to the ends of the DNA, followed by enrichment of tagged DNA with Streptavidin beads. DNA is denatured and a single-strand break (SSB) is generated by alkaline-cleavage conditions at the abasic site. A second adapter is ligated at the abasic site position and the NGS library is generated by PCR and sequenced using for example an ILLUMINA MiSeq. Alignment of sequences against the reference genome reveals the location of the abasic site positions where enrichment is found. The original 5hmC position corresponds to the base located before the start of said enriched reads. A representative approach that can be adapted to be used with the described Brig2 protein as described in this example is described in Liu, Z.J., Martínez Cuesta, S., van Delft, P. et al. Sequencing abasic sites in DNA at single-nucleotide resolution. Nat. Chem.11, 629–637 (2019), the disclosure of which is incorporated herein by reference.2. SSiNGLe-AP:In this example, genomic DNA is fragmented and denatured. Abasic sites are generated at the 5hmC nucleobase positions using Brig2. As a control, genomic DNA with no Brig2 treatment is used. The endogenous 3’OH termini of the fragments are blocked with terminal transferase and biotin-11-dCTP. The blocked DNA is captured using streptavidin- coated magnetic beads and treated with an enzyme that generates SSBs at the abasic sites. The newly created free 3’OH termini are tagged by polyA-tailing that is used for NGS library preparation. Paired-end sequencing is carried out with ILLUMINA NovaSeq. Abasic sites are located by shifting the position of the first aligned base, which corresponds to the position of the enzyme-generated 3’OH, by one base. A representative approach that can be adapted to be used with a described Brig2 protein as described in this example is described in Abasic site position determines original 5hmC position in the genome. Cai, Y., Cao, H., Wang, F. et al. Complex genomic patterns of abasic sites in mammalian DNA revealed by a high- resolution SSiNGLe-AP method. Nat Commun 13, 5868 (2022), the disclosure of which is incorporated herein by reference. In an example, use of a synthetic probe for enrichment of DNA fragments with abasic sites can be bypassed by using a described catalytically dead Brig2 (dBrig2) with a His or other suitable purification tag, which can bind 5hmC but not cleave the nucleobase. Incubation of DNA fragments with dBrig2 and treatment with Ni-NTA magnetic beads will select for fragments with 5hmC residues which can then be treated with active Brig2 to generate abasic sites for sequencing. This can be coupled with any of the above-mentioned library preparation and sequencing techniques. A dBrig2 mutant can also be harnessed as a binding partner to detect and bind 5hmC.3. TLS-APseq:In this example, genomic DNA is fragmented and different adapters are ligated to the 5’ and 3’ termini of the fragments followed by Brig2 treatment to generate abasic sites. A control DNA library is left untreated. Extension of fragments with abasic sites is performed using primers complementary to the 5’ and 3’ adapters and the translesion synthesis (TLS) polymerase, Sulfolobus DNA polymerase IV, which is commercially available. This polymerase is able to replicate DNA past the abasic site by adding a nucleobase, typically adenine across the abasic site. A high-fidelity DNA Polymerase is added in a 1:1 ratio to make extension of the full fragments possible. This is expected to yield a DNA molecule with an A-T pairing at the abasic site position. Amplification of the library by PCR with the repaired DNA is performed as indicated by the NebNext Ultra FS II protocol and its indices for library preparation. Paired-end sequencing is carried out with ILLUMINA MiSeq or ILLUMINA NovaSeq. Sequencing reads are mapped back to the reference genome and only “errors” that are found in every copy or substantially all copies are regarded as an abasic site, with a change from C to A if the leading strand is being read, or from G to T if the lagging strand is being read. A non-limiting demonstration of TSL-AP seq is shown by FIG. 10. In this approach. As discussed above, TLS-AP seq is used to sequence 5hmC in DNA. This method relates in part to NGS for the Illumina platform by integrating steps of the NEBNext Ultra II FS DNA Library Prep Kit and using T2 as the model DNA for 5hmC detection at a single-nucleotide resolution. The method is depicted in FIG. 10(A). First, Brig2 is used to generate abasic sites in DNA where 5hmC bases are found. Abasic sites cannot be sequenced with standard NGS library prep procedures due to the instability of the DNA with abasic sites, and because NGS is based on PCR amplification which is not possible with a replicative polymerase on DNA with abasic sites. The disclosure demonstrates a means to achieve amplification of this DNA with abasic sites by regenerating the DNA, and inserting specific nucleotides into the abasic sites positions. Trans-lession synthesis (TLS) polymerases are able to repair DNA with abasic sites by inserting an Adenine (A) opposite to the abasic site, however these polymerases are not replicative and only extend past DNA lesions by 1 or 2 nucleotides As such, the disclosure instead uses a mix of Taq Polymerase and a TLS Polymerase, Sulfolobus DNA Pol V, to amplify the DNA with abasic sites generated by Brig2. This reconstituted DNA copy will contain an “A” where the original 5hmC was found, which can be subsequently identified by mapping the sequencing “errors” between an untreated control and a sample treated with Brig2. This allows for single-nucleotide resolution mapping of 5hmC bases in a genome. The disclosure demonstrates integration of these principles into a single step that is added to the NEBNext Ultra II FS DNA Library Prep workflow. The proof of principle for this method is shown with T2 DNA, which contains 5hmC and glucosylated 5hmC. TLS-AP Seq is shown to able to detect the non glucosylated 5hmC bases in the T2 DNA using the following representatives steps” 1. Genomic DNA is fragmented to approximately 200 bp fragments enzymatically following thekit’s instructions. 2. NEB Next adaptors are ligated to the DNA fragments.Cleaning step with beads. 3. DNA with adaptors is subject to the TLS-APseq enzymatic treatment:A. Initial denaturation of DNA at 95CB. Annealing of primers complementary to the adaptors at 63CC. Addition of Brig2 and incubation for 30 minutes at 37C.D. Addition of Taq Polymerase and dNTPs and incubation for 10 minutes at 37C.E. Addition of Pol IV and incubation for 1 hour at 37C.F. TLS-AP seq PCR cycle (15 cycles):^ Denaturation for 30 seconds at 85C^ Primer annealing for 30 seconds at 63C^ Extension for 3 minutes at 68C.^ Final extension for 5 minutes at 68C.Cleaning step with beads. 4. Index PCR of the amplified fragments of DNA.Cleaning step with beads. 5. Next Generation Sequencing with Illumina Miseq6. Genome assembly and data processing with TLS-Ap seq code.For steps 3C, 3D and 3E the enzymes and dNTPs are added manually to the PCR tube while the reaction is still in the thermocycler. Representative results from the described process are shown in FIG 10. These results were obtained by sequencing four T2 DNA samples, two were untreated controls and two were treated with Brig2. In the untreated controls, one sample did not undergo the TLS-AP seq PCR cycle, the extra step added to the NEB workflow, while the other did. This was to account for the errors that could be accumulated as background from using the Taq and Pol IV mix for DNA reconstitution prior to library amplification with the NGS indexes. The statistics showed that only the treated samples (3 and 4) had a significant amount of mutations on C or G bases that were read as A or T (depending if the forward or lagging strand were being read) (FIG.10 (B). This shows the removal of 5hmC from the DNA by Brig2 and reconstitution of the damaged DNA by Pol V integrating an A opposite of the abasic site. The example also demonstrates locating every mutated base in the genome, compared to the reference T2 genome. These results confirm that the described TLS-AP seq functions to detect 5hmC in DNA at a single-nucleotide resolution. A comparison from a kit available from NEB for NGS library preparation to specifically detect 5hmC involves more steps and reagents, and the process is more laborious than TLS-AP seq (FIG.10C). Nanopore Sequencing: This example relates to a crown-ether electrolyte adduct. In this example, genomic DNA is extracted. Abasic sites are generated at the 5hmC nucleobase positions using Brig2. As a control, genomic DNA with no Brig2 treatment is used. The DNA is incubated with 2- aminomethyl-18-crown-6 in the presence of NaBH3CN.2-aminomethyl-18-crown-6 is functionalized to the abasic sites via reductive amination forming a stable adduct. This adduct generates unique amplitude current signatures when translocated through a nanopore. DNA fragments are sequenced using the MinION from Oxford Nanopore. Base-calling software is modified to recognize the unique electrical signature generated by the adduct as an abasic site. A representative approach that can be adapted to be used with a described Brig2 protein as described in this example is described in An N, Fleming AM, White HS, Burrows CJ. Crown ether-electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules. Proc Natl Acad Sci U S A.2012 Jul 17;109(29):11504-9. doi: 10.1073 / pnas.1201669109. Epub 2012 Jun 1, the disclosure of which is incorporated herein by reference. In examples, the disclosure provides an article of manufacture, which may comprise a kit. In examples, the article of manufacture may comprise a described Brig2 protein, and one or reagents for use in the described methods. An article of manufacture may include one or more sealed containers that contain any of the aforementioned components, and may further comprise packaging and / or printed material. The printed material may provide information on the contents of the article, and may provide instructions or other indication of how the contents of the article can be used, such as for isolating, immobilizing, detecting, or sequencing a DNA sample. The disclosure uses bacteriophages (i.e., phages) to reveal the Brig2 protein as discussed above, and its catalytic activity, as follows. Phages encompass a diverse group of bacteria-infecting viruses which shape bacterial communities by applying constant selective pressure. To survive phage infection, bacteria have evolved diverse and sophisticated immune systems that target different stages of the phage lifecycle as well as their structural features and nucleic acids. Restriction-modification (R-M) systems are a prominent innate immune mechanism that rely on restriction endonucleases as the main effector of antiviral immunity. These enzymes recognize DNA motifs present in host and viral genomes and cleave DNA in a sequence-specific manner. To prevent autoimmunity and self-cleavage by restriction endonucleases, bacteria have developed a mechanism for self / non-self-differentiation through methylation of its own genome, thereby enabling cleavage of only unmethylated viral DNA. Some phages counteract bacterial restriction strategies by introducing DNA nucleobase modifications into their own genomes. For example, the lytic bacteriophage T4, which infects E. coli, contains 5-hydroxymethylcytosine (5hmC) instead of cytosine in its DNA, and encodes glucosyltransferases that glycosylate 5hmC to glucosyl-5hmC. These “hypermodified” nucleobases protect the viral genomic DNA against anti-phage defense mechanisms such as types I, II, and III restriction endonucleases and even CRISPR-Cas. The T4-related T-even family phages T2 and T6 also contain hypermodifications of 5hmC in their DNA. While T4 contains fully glucosylated 5hmC, with ~70% in ^-linkages (introduced by T4 alpha- glucosyltransferase, a-gt) and ~30% in ^-linkages (introduced by T4 beta- glucosyltransferase, b-gt), T2 and T6 lack b-gt and hence have no ^-linked glucose modifications. Instead, T2 has only 75% of its 5hmC glucosylated, with ~70% in ^-linkages and the remaining ~5% modified with an additional second glucose, ^-linked to the primary glucose, to give gentiobiosyl-5hmC. T6, in contrast, has ~72% of its 5hmC sites doubly glucosylated as gentiobiosyl-5hmC and only ~3% in the form of ^-glucosyl-5hmC. To counter these DNA modifications in T-even phages, E. coli has evolved specialized restriction enzymes, known as type IV enzymes, that can cleave 5hmC- or glucosylated 5hmC-containing DNA. Examples include McrBC, which cleaves 5-methylcytosine (5mC) and 5hmC-containing DNA, and GmrSD, which cleaves glucosylated 5hmC-containing DNA carried by T-even phages. This disclosure involves infection of soil metagenomic library with a mutant T4 phage that lacks DNA glycosylation, thereby revealing the described Brig2 DNA glycosylase, that targets 5hmC nucleobases to provide anti-phage immunity against T-even phages that widely carry these DNA bases. This discovery facilitated development of Brig2 (and its catalytically dead derivative) for use in the methods described above. The identification of Brig2 and dBrig2 is illustrated by the following results. Infection of E. coli harboring an environmental DNA library uncovers a gene that targets 5-hydroxymethylcytosine nucleobases We performed a metagenomic environmental DNA (eDNA) screen directed towards the identification of an anti-phage defense system that recognizes 5hmC nucleobases (Fig. 5A). To do this, we first engineered a T4 phage that lacks the alpha- and beta- glucosyltransferase genes (T4 Δa-gt Δb-gt), yielding a phage without DNA glycosylation and carrying only unmodified 5hmC nucleobases in its genome. The Arizona soil eDNA library, AZ52 was used for the functional selection. This library is composed of E. coli EC100 cells transformed with millions of different cosmids, each containing a ~40 kb DNA insert from a soil-dwelling organism. The AZ52 library was infected with T4 Δa-gt Δb-gt at a high multiplicity of infection (MOI 10) and DNA from surviving cosmids was re-transformed into a fresh batch of E. coli EC100 cells to give a phage-enriched library. Fourteen individual colonies from this library were randomly selected and challenged with T4 Δa-gt Δb-gt or T5, as a control, in LB top agar plaque assays. T5 serves as a control since this phage does not belong to the T-even family and lacks 5hmC nucleobases. We found two colonies, clones A and B, that carried immunity to T4 Δa-gt Δb-gt but not to T5 (Fig.5A) Specifically, clone A exhibited up to 5-logfold reduction in plaque forming units (PFUs) (Fig.5A). Next- generation sequencing (NGS) of the cosmid from clone A revealed that its metagenomic DNA insert harbors a bona fide bacterial defense island (Fig.5B), including a type II-E CBASS22system and genes belonging to a SspABCD-SspE defense system. To identify the genes responsible for T4 Δa-gt Δb-gt immunity, we divided the fragment into six different subclones, 1-6, and found that subclone 6 retained the selected phenotype (Fig.5C). This fragment contained three genes, gene 1 with homology to an unknown protein belonging to the superfamily of uracil DNA glycosylases gene 2 with unknown structure and function, and gene 3 predicted to have ADP-ribosyl glycohydrolase activity. Genes 1 and 2 were in the same operon. Genetic dissection of the defense fragment into 4 different subfragments, 1-4, demonstrated that gene 1 solely, present in pFragment6-1 and pFragment6-4, was responsible for the immunity of E. coli hosts to T4 Δa-gt Δb-gt (Fig. 1A). These subfragments also provided strong defense against T-even phages T2 and T6, and to a mutant T6 phage, T6 escaper 1, which carries a mutation in its a-gt gene predicted to eliminate DNA glucosylation (Fig.1B). While T2 and T6 phages have both alpha-glucosyl- and gentiobiosyl-5hmC nucleobases in their genomes, a large fraction of their 5hmC nucleobases always remain without glucosylation (~25%). T6 escaper 1 harbors non- glucosylated 5hmC nucleobases akin to T4 Δa-gt Δb-gt. Our results therefore indicate that genomes with at least some presence of 5hmC nucleobases are susceptible to immunity mediated by gene 1. Moreover, expression of gene 1 using an arabinose-inducible promoter showed immunity only in the presence of the transcription inducer (Fig.1C), a result which demonstrates that gene 1 drives the observed immunity against T4 Δa-gt Δb-gt. To investigate the function of the operon bearing genes 1 and 2, stop codons were inserted into each of these genes (Fig.1D). A stop codon in gene 1 fully eliminated T4 Δa-gt Δb-gt immunity, even when expressed with gene 2. This confirms that gene 1 is the driver of T4 Δa-gt Δb-gt immunity. Insertion of a stop codon into gene 2 eliminated operon-mediated defense against wild-type T4 phage (Fig.1D). Since defense against T4 is only observed when both genes are expressed (Fig.1A, D), the disclosure analyzed whether a genetic interaction between the two genes which facilitates immunity against phage T4, the only phage tested that contains fully glucosylated 5hmC nucleobases genome-wide. Altogether, these experiments demonstrate that the DNA glycosylase encoded by gene 1 prevents the replication of T4 Δa-gt Δb-gt viral DNA, and therefore we named this gene bacteriophage replication inhibition DNA glycosylase 2, Brig2. The plasmid harboring only gene 1, was renamed pBrig2. Gene 2 was renamed BapA, for Brig-associated protein A, given its functionality and association with Brig2. The plasmid harboring both genes was renamed pBrig2-BapA. Brig2 provides immunity against phages whose genomes contain 5- hydroxymethylcytosine nucleobases The antiviral activity of Brig2 against phage T4 Δa-gt Δb-gt was confirmed in liquid culture (Fig.6A-C), as well as the cooperative defense mediated by Brig2 and BapA against wild-type T4 (Fig.6A, D-E). Anti-phage defense provided by Brig2 was also confirmed in liquid culture for phages T2 (Fig.7A-C) and T6 (Fig.7A, D-F), however, there were no detectable differences in defense when Brig2 was co-expressed with BapA (Fig.7). Brig2 was also tested against 11 other 5hmC-containing T-even phages from the BASEL phage collection (Bas35 to Bas45) (Fig.2A). Brig2 provided strong defense against all tested phages, confirming that this DNA glycosylase targets phages with 5hmC bases in their genomes. To confirm that Brig2 can only target 5hmC bases that are not glucosylated, we carried out heterologous expression of a-gt and b-gt. Plasmid-borne expression of b-gt protected phage T4 Δa-gt Δb-gt from Brig2 targeting when expressed on its own or alongside BapA (Fig.2B). These results were confirmed by complementation of b-gt in a T4 mutantphage that lacks only this glucosyltransferase (T4 Δb-gt). Expression of b-gt rescued theability of the mutant phage to form plaques and overcome Brig2-mediated immunity (Fig. 2C). Hence, glucosylation of 5hmC bases by b-gt prevents Brig2 activity. Overexpression of a-gt, however, did not provide the same effect (Fig.2B). We hypothesize that this is due to the ability of beta-glucosyltransferase to glucosylate adjacent 5hmC nucleobases, achieving full genome-wide 5hmC glucosylation, while a-gt is unable to glucosylate side-by-side 5hmC nucleobases, enabling glucosylation of only ~75% of the 5hmC nucleobases genome-wide. These results are in line with the presence of non-glucosylated 5hmC nucleobases in T-even phage genomes such as T2 and T6 which express a-gt and lack b-gt and are accordingly sensitive to Brig2 targeting. On the other hand, phage T4, which encodes both a-gt and b-gt, has full genome-wide 5hmC glucosylation, enabling resistance to Brig-2 mediated immunity in the absence of BapA (Fig.1A, D). Brig2 is a DNA glycosylase that excises 5hmC nucleobases to generates abasic sites in DNA We performed an Alphafold2 protein structure prediction of Brig2 which yielded the high confidence structural model shown in Fig.8A and predicted structural homologs of the protein using the DALI server. The first hit was a uracil DNA glycosylase from the bacterium Thermus thermophilus that excises uracil and hydroxymethyluracil from DNA. Uracil DNA glycosylases hydrolyze the glycosidic bond between the base and the sugar. This reaction is facilitated by flipping the uracil base into a catalytic pocket within the enzyme. The generation of an abasic site at the position of the uracil initiates base excision repair. We analyzed whether Brig2 acts by excising 5hmC nucleobases since it provides defense only against phages that contain some fraction of non-glucosylated 5hmC. To do this , we incubated Brig2 with a 60 bp DNA oligonucleotide containing a single modified nucleobase, followed by treatment with heat and NaOH to cleave abasic sites site-specifically by beta- elimination to give lower molecular weight DNA that can be detected by gel electrophoresis. Cleavage was observed for oligonucleotide substrates containing 5hmC and uracil, only in the presence of heat and NaOH, demonstrating that Brig2 excises both 5hmC and uracil nucleobases (Fig.3A, B). The presence of uracil base excision suggests that Brig2 evolved from homologous uracil DNA glycosylases. Additional experiments showed that Brig2 is highly active on 5hmC, generating abasic sites at concentrations as low as 1 nM (Fig.3C, D). To test if the affinity of Brig2 is greater for 5hmC than for uracil, we incubated the 5hmC- and uracil-containing oligonucleotides with a range of concentrations of the enzyme (Fig.3E) and estimated a Kmvalue for each substrate, using the upper band intensity values obtained using FIJI (Fig.3F). The Km for 5hmC is significantly lower (between 1 and 10 nM) than it is for uracil substrate (Kmbetween 100 and 1000 nM), which confirms that the affinity of Brig2 is substantially higher for the 5hmC substrate. These results, coupled with its presence in a bacterial defense island, suggest that Brig2 evolved divergently from canonical members of the uracil DNA glycosylase superfamily to specifically target 5hmC in the context of anti- phage defense. The nucleotide binding pocket is predicted as shown (Fig.8B, C). To test if the putative binding pocket is important for Brig2 activity, we mutated amino acids predicted to outline this area: Y97 and D127 (Fig.8B). We found that while the Y97A substitution did not substantially affect Brig2-mediated immunity, the D127A mutant failed to reduce T4 Δa-gt Δb-gt plaque formation (Fig.8D). Substitution of D127 for asparagine, which eliminates the negative charge on this residue, also abrogated Brig2 activity (Fig.8D). Substitution of Y97 for phenylalanine reduced the anti-phage activity of Brig2 albeit to a lesser extent than the D127 mutations (Fig.8D). These results demonstrate that the DNA glycosylase catalytic pocket of Brig2 is necessary for base excision activity and defense against phage T4 Δa-gt Δb-gt, but is dispensable for use of Brig2 as a binding agent, as discussed above.Brig2 degrades T4 Δa-gt Δb-gt DNA in vitroTo investigate the molecular mechanism by which Brig2 restricts T4 Δa-gt Δb-gt infection, we tested the effect of purified Brig2 on phage DNA in vitro. We incubated wild- type and T4 Δa-gt Δb-gt phage DNA with Brig2 for 30 minutes at 37°C and visualized the products via agarose gel electrophoresis (Fig.4A). A second set of samples was incubated for an extra 20 minutes at 65°C to cleave abasic sites via beta-elimination (Fig.4B). We observed full degradation of T4 Δa-gt Δb-gt DNA even at the lowest concentration of Brig2 used (2Nm) and before heat treatment (Fig.4A, B). This suggests that the enzyme is highly active on phage DNA that contains 5hmC. We did not observe degradation of wild type phage or plasmid DNA. We did, however, observe a mobility shift for wild type T4 DNA as we increased the concentration of Brig2. We attribute this non-specific DNA binding to the ability of Brig2 to bind non-target DNA and perform target search, as has been hypothesized for uracil DNA glycosylases. In addition, we incubated Brig2 with T4 Δa-gt and T4 Δb-gt DNA to assess if we would observe reduced degradation of DNA that contains a lower fraction of non-glucosylated 5hmC nucleobases (Fig.4C, D) compared to the fully non- glucosylated double mutant (Fig.4A, B). Incubation of Brig2 with T4 Δa-gt and T4 Δa-gt DNA revealed less degradation than the double mutant in both cases and comparable to wild- type T4 DNA (Fig.4A, C). Altogether these data indicate that Brig2 generates abasic sites and promotes beta-elimination-mediated degradation of phage DNA that contains 5hmC bases that are not glucosylated. It will be apparent from the foregoing that Brig2 is a new DNA glycosylase that provides antiviral defense against phages that carry DNA modifications. Brig2 is adjacent to an ADP-ribosyl glycohydrolase protein, suggesting that these proteins regulate Brig activity and / or remove deactivating ADP-ribose modifications of Brig proteins. Brig2 targets 5hmC nucleobases, a widespread DNA modification found across phages including all known members of the T-even family phages that infect E. coli. Data on ssDNA oligonucleotides and phage-extracted dsDNA indicate that Brig2 acts on both ssDNA and dsDNA substrates. Given its ability to excise 5hmC nucleobases, Brig2 is suitable for use to detect and sequence 5hmC nucleobases across prokaryotic and eukaryotic genomes, including the human genome where 5hmC is a common DNA mark in neurons and embryonic stem cells that can regulate transcription, as discussed above. While the disclosure has been particularly shown and described with reference to specific examples, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

What is claimed is:

1. An isolated protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”).

2. The isolated Brig2 protein of claim 1, comprising a change in amino acid position in SEQ ID NO:1 at position 127 (dBrig2), wherein the change is to an amino acid other than D, and wherein the Brig2 protein comprising the amino acid change has reduced or eliminated capability to excise 5-hydroxymethylcytosine (5hmC) nucleobases to generate abasic sites.

3. The isolated protein of claim 1 or claim 2, further comprising an addition of amino acids, wherein the addition of amino acids optionally comprises a purification tag.

4. The isolated protein of claim 1 or claim 2, further comprising a detectable label or wherein the isolated protein is present in a fusion protein with a detectable protein.

5. The isolated protein of claim 1 or claim 2, wherein the isolated protein is attached to a solid substrate and / or is present in a complex with a 5-hydroxymethylcytosine nucleobase that is present on a DNA polynucleotide.

6. A method comprising contacting a DNA sample with a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”) to generate abasic sites at 5-hydroxymethylcytosine (5hmC) nucleobases with the DNA sample.

7. The method of claim 6, further comprising determining the location of the abasic sites in the DNA sample.

8. The method of claim 6 or claim 7, wherein the DNA sample is contacted with a dBrig2 protein to bind to 5-hydroxymethylcytosine nucleobases, prior to determining the location of abasic sites in the DNA sample.

9. A kit comprising a sealed container and a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”).

10. The kit of claim 9, further comprising one or more sealed containers that contain reagents for use of the protein for determining the presence and / or location of 5- hydroxymethylcytosine nucleobases in a DNA sample.

11. An isolated polynucleotide encoding a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”).

12. The isolated polynucleotide of claim 11, wherein the isolated polynucleotide is present in an expression vector.

13. A method comprising allowing expression of a protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”) from an expression vector introduced into a population of cells in vitro.

14. The method of claim 13, further comprising separating the Brig2 from the population of cells.

15. A protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:1 (“Brig2”) that has been isolated from the population of cells of claim 14.

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