Distinct structural and functional heterochromatin partitioning of lamin B1 and lamin B2 revealed using genome-wide nicking enzyme epitope targeted DNA sequencing.

Gene expression is regulated by chromatin DNA methylation and other features, including histone post-translational modifications (PTMs), chromatin remodelers and transcription factor occupancy. A complete understanding of gene regulation will require the mapping of these chromatin features in small cell number samples. Here we describe a novel genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq).

Structural analysis of the BisI family of modification dependent restriction endonucleases.

The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA.

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP.

High-throughput sequencing of EcoWI restriction fragments maps the genome-wide landscape of phosphorothioate modification at base resolution.

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genomes and their exact roles in the cell remain to be defined. In this study, we developed a simple and convenient technique called EcoWI-seq based on a modification-dependent restriction endonuclease to identify genomic positions of PT modifications.

Expression and Purification of XI Restriction Endonuclease and Engineering New Specificity From XI Specificity Subunit.

It is stated that XI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5'↓N9 AC N5 CTCC N10↓ 3' (complement strand 5' ↓N7 GGAG N5 GT N12↓ 3'), creating 3-base 3' overhangs. Here we report the cloning and expression of and genes in . The XI activity was successfully reconstituted by mixing the XI RM fusion subunit with the XI S subunit and the enzyme complex further purified by chromatography over 6 columns.

Engineering Infrequent DNA Nicking Endonuclease by Fusion of a HI Cleavage-Deficient Mutant and a DNA Nicking Domain.

Strand-specific DNA nicking endonucleases (NEases) typically nick 3-7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A HI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from HI restriction.

Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, Ribonuclease I and Human RNase A.

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome .

Mechanism of DNA cleavage by the endonuclease SauUSI: a major barrier to horizontal gene transfer and antibiotic resistance in Staphylococcus aureus.

Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus.

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA.

Type II Restriction of Bacteriophage DNA With Modified Pyrimidines.

To counteract host-encoded restriction systems, bacteriophages (phages) incorporate modified bases in their genomes. For example, phages carry in their genomes modified pyrimidines such as 5-hydroxymethyl-cytosine (5hmC) in T4 deficient in α- and β-glycosyltransferases, glucosylated-5-hydroxymethylcytosine (5gmC) in T4, 5-methylcytosine (5mC) in Xp12, and 5-hydroxymethyldeoxyuridine (5hmdU) in SP8. In this work we sequenced phage Xp12 and SP8 genomes and examined Type II restriction of T4, T4, Xp12, and SP8 phage DNAs.

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases.

Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2-4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA.

Universal NicE-seq for high-resolution accessible chromatin profiling for formaldehyde-fixed and FFPE tissues.

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues.

A protein architecture guided screen for modification dependent restriction endonucleases.

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups.

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications.

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: phage M6 with 5-(2-aminoethyl)deoxyuridine (5-edU), phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-emdU) and phage phi W-14 (a.k.

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI.

TagI belongs to the recently characterized SRA-HNH family of modification-dependent restriction endonucleases (REases) that also includes ScoA3IV (Sco5333) and TbiR51I (Tbis1). Here, we present a crystal structure of dimeric TagI, which exhibits a DNA binding site formed jointly by the nuclease domains, and separate binding sites for modified DNA bases in the two protomers. The nuclease domains have characteristic features of HNH/ββα-Me REases, and catalyze nicks or double strand breaks, with preference for /RY and RYN/RY sites, respectively.

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease.

Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease.

Restriction and modification of deoxyarchaeosine (dG)-containing phage 9 g DNA.

E. coli phage 9 g contains the modified base deoxyarchaeosine (dG) in its genome. The phage encodes its own primase, DNA ligase, DNA polymerase, and enzymes necessary to synthesize and incorporate dG.

NicE-seq: high resolution open chromatin profiling.

Open chromatin profiling integrates information across diverse regulatory elements to reveal the transcriptionally active genome. Tn5 transposase and DNase I sequencing-based methods prefer native or high cell numbers. Here, we describe NicE-seq (nicking enzyme assisted sequencing) for high-resolution open chromatin profiling on both native and formaldehyde-fixed cells.

Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA.

Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms.

Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes.

The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates.

Sequence-specific DNA nicking endonucleases.

A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma.