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.

The oncoprotein Mdm2, and the recently intensely studied, homologues protein Mdmx, are principal negative regulators of the p53 tumor suppressor. The mechanisms by which they regulate the stability and activity of p53 are not fully established. We have determined the crystal structure of the N-terminal domain of Mdmx bound to a 15-residue p53 peptide. The structure reveals that although the principle features of the Mdm2-p53 interaction are preserved in the Mdmx-p53 complex, the Mdmx hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by sidechains of Met and Tyr of the p53-binding pocket of Mdmx. Thus specific inhibitors of Mdm2-p53 would not be optimal for binding to Mdmx. Our binding assays show indeed that nutlins, the newly discovered, potent antagonists of the Mdm2-p53 interaction, are not capable to efficiently disrupt the Mdmx-p53 interaction. To achieve full activation of p53 in tumor cells, compounds that are specific for Mdmx are necessary to complement the Mdm2 specific binders.

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.

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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 5methylcytosine (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.

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 fourstranded ␤-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded ␤-sheets. Moreover, the ␤-sheets in Int HPI and Int HAI7 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.Genomic islands, together with temperate phages, integrative plasmids, transposons, and integrative conjugative elements, make up the group of mobile genetic elements that play an important role in bacterial quantum leap evolution and adaptation (1-3). Genomic islands that carry clustered genes encoding vital functions supply bacteria with additional capabilities to withstand and overcome host defenses and to improve fitness. Genomic islands are integrative elements that are not able to self-transfer and replicate. Typically, they are composed of functional and recombination modules. The recombination module consists of a tyrosine family integrase and two attachment sites involved in recombination. The integrase promotes attPϫ attB site-specific DNA recombination of the genomic islands into highly conserved tRNA-encoding genes (attB recombination targets) of the host genome and subsequent excision (4,5).It has been demonstrated that the N-terminal domain of phage integrases is responsible for specific recognition of the arm-type site sequence of the attachment sites, a step that is essential for activity of the catalytic C-terminal domain responsible for the strand exchange. Three-dimensional structures of the N-terminal domains of two prokaryotic integrases, namely bacteriophage integrase and Tn916 transposon integrase, have been determined. Although they do not share significant sequence homology, both adopt similar structures and recognize the arm-type DNA site by inserting their N-terminal domain into a major groove of DNA (6 -9). The N-terminal domain, consisting of a three-stranded antiparallel ␤-sheet, is proposed to be a new DNA-binding motif whose residue composition and position within the major DNA groove varied to alter specificity (6). Nevertheless, the genomic islands are evolutionarily divergent from phages and other mobile elements and represent a distinct mobile genetic element class. Moreover, island-encoded integrases are not closely related to phage integrases, as was expected previously (3, 10).Four closely related genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin b...

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