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

Abstract

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.

Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction

“…First, single cells are sorted into 384-well plates and, adopting a previously published method for bulk 5hmC sequencing 7 , 5hmC marks are glucosylated using T4 phage β-glucosyltransferase. Next, this glucosylated form of 5hmC is recognized by AbaSI, which generates a double-stranded break with a 2-nucleotide overhang, 11-13 bp downstream of its binding site 7,19 . Subsequently, the digested gDNA is ligated to double-stranded adapters containing a 2-nucleotide random 3′ overhang, together with a cell-specific barcode, the Illumina 5′ adaptor and a T7 promoter.…”

DNA Base Flipping: A General Mechanism for Writing, Reading, and Erasing DNA Modifications

“…AbaSI, unlike MspJI, has an N-terminal Vsr-like endonuclease domain and a C-terminal SRA-like domain (Borgaro and Zhu 2013; Horton et al 2014a). Its SRA domain seems to preferentially recognize 5ghmC and 5hmC compared to 5mC, as the relative rate of cleavage of DNA containing the corresponding modification is 5ghmC:5hmC:5mC = 8000:500:1 (Wang et al 2011).…”

“…Mammalian and plant SET- and RING-associated (SRA) domains recognize 5mC within the genome by base flipping (Arita et al 2008; Avvakumov et al 2008; Hashimoto et al 2008; Rajakumara et al 2011) and have been characterized as nonenzymatic base flippers. Since the first discovery in eukaryotes, SRA domains have been rediscovered in prokaryotes, recognizing 5mC, 5hmC, and/or 5ghmC to coordinate restriction activity in a modification-dependent manner (Horton et al 2012, 2014a, b, c). In addition to SRA, the bacterial modified cytosine restriction B enzyme also flips 5mC for recognitions but is structurally distinct from other known base flippers (Sukackaite et al 2012).…”

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

“…There is much interest in understanding where these forms occur in genomes, how they differ between cell types and change with time and circumstances and how they affect gene expression. Recently discovered ‘modification-dependent’ restriction enzymes such as MspJI/AspBHI ( 8 , 9 ), and PvuRts1I/AbaSI ( 10 , 11 ), provide a way of answering some of these questions at single-base resolution. These enzymes bind to duplex DNA at sequences containing certain modifications and cleave the DNA a short distance away, generating fragments that can be sequenced and analyzed by bioinformatics ( 7 , 12 , 13 ).…”