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

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ( hm C), is generated by enzymatic oxidation of 5-methylcytosine ( m C). This discovery has raised fundamental questions about the functional relevance of hm C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare hm C against the far more abundant m C is technically challenging and to date no methods for direct sequencing of hm C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hm C-containing sequences. We determined the consensus cleavage site of PvuRts1I as hm CN 11–12 /N 9–10 G and show first data on its potential to interrogate hm C patterns in mammalian genomes

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes

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

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

Structures of the Arm-type Binding Domains of HPI and HAI7 Integrases*

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel four-stranded β-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded β-sheets. Moreover, the β-sheets in IntHPI and IntHAI7 are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism