Aine Quimby

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3′-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11–13 nt away in the top strand and 9–10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′, with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.

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.

Characterization of cleavage intermediate and star sites of RM.Tth111II

Tth111II is a thermostable Type IIGS restriction enzyme that recognizes DNA sites CAARCA (R = A or G) and cleaves downstream at N11/N9. Here, the tth111IIRM gene was cloned and expressed in E. coli, and Tth111II was purified. The purified enzyme contains internally-bound S-adenosylmethionine (SAM). When the internal SAM was removed, the endonuclease activity was stimulated by adding SAM or its analog sinefungin. The cleavage intermediate is mostly top-strand nicked DNA on a single-site plasmid. Addition of duplex oligos with a cognate site stimulates cleavage activity of the one-site substrate. Tth111II cleaves a two-site plasmid DNA with equal efficiency regardless of site orientation. We propose the top-strand nicking is carried out by a Tth111II monomer and bottom-strand cleavage is carried out by a transient dimer. Tth111II methylates cleavage product-like duplex oligos CAAACAN9, but the modification rate is estimated to be much slower than the top-strand nicking rate. We cloned and sequenced a number of Tth111II star sites which are 1-bp different from the cognate sites. A biochemical pathway is proposed for the restriction and methylation activities of Tth111II.