Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5–hydroxymethylcytosine

Abstract

In mammalian genomes a sixth base, 5-hydroxymethylcytosine (hmC), is generated by enzymatic oxidation of 5-methylcytosine (mC). This discovery has raised fundamental questions about the functional relevance of hmC in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare hmC against the far more abundant mC is technically challenging and to date no methods for direct sequencing of hmC have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hmC-containing sequences. We determined the consensus cleavage site of PvuRts1I as hmCN11–12/N9–10G and show first data on its potential to interrogate hmC patterns in mammalian genomes.

Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood

“…The PvuRts1i enzyme cuts 12-15 nucleotides away from the 5hmC sites. 33 Therefore, regions which show significantly higher coverage compared to the surrounding noise levels (also called peaks) are indicative of the presence of 5hmC within § 12-15 nucleotides from the genomic location of the peaks. 25 We found that Pvu-Seq can only validate the presence or absence of 5hmC site (s) and cannot be used quantitatively at the sequencing depth that we used in this paper.…”

“…69 DNA digestion with PvuRst1L and DNA sequencing PvuRts1I (Pvu) restriction enzyme can directly cleave hydroxymethylated DNA 12-14 bps away from the 5hmC site. 33 We developed a new technique that we call Pvu-seq which allows direct detection of 5hmC without chemical modification of 5hmC or bisulfite conversion. A paper describing this technique was published in BMC Genomics.…”

“…For our study the estimated coverage cutoff was 4.5. PvuRts1I (Pvu) restriction enzyme can directly cleave hydroxymethylated DNA 12-14 bps away from the 5hmC site 33 sites. Therefore we extended our peak region to §20 on either side.…”

5‐Hydroxymethylcytosine, the Sixth Base of the Genome

“…[32] Furthermore, the endonuclease PvuRts1l has been identified to specifically act on hmC. [33] In the future, nanopore sequencing might be a solution to the problem. First studies showed that this sequencing method is indeed able to distinguish between mC and hmC in both single-and double-stranded DNA.…”

5‐Hydroxymethylcytosin, die sechste Base des Genoms

“…[32] Zusätzlich wurde die Endonuklease PvuRts1l identifiziert, die spezifisch auf hmC wirkt. [33] In Zukunft könnte Nanoporensequenzierung eine Lösung für dieses Problem sein. Erste Untersuchungen zeigten, dass diese Sequenzierungsmethode zwischen mC und hmC in Einzel-und Doppelstrang-DNA unterscheiden kann.…”