Post-doc, University of New Mexico
Vertebrate Endonuclease G evolved to recognize 5-hydroxymethylcytosine for DNA recombination
Hydroxymethylcytosine (5hmC) is a modified version of cytosine (C), one of the 4 letters of the genetic code. 5hmC was recently discovered to be naturally present in up to 1% of C's in the mammalian genome, and it is only present in vertebrate DNA. We have discovered that Endonuclease G (EndoG), a DNA binding protein, has evolved in vertebrate species to be able to distinguish 5hmC from C. Not many proteins can distinguish C from 5hmC, so EndoG likely has an important function related to 5hmC. We hypothesize that 5hmC marks specific DNA sites to promote DNA recombination, a process used to repair damaged DNA. Overall we study how 5hmC changes DNA structure. We want to know how EndoG is able to specifically bind to 5hmC DNA, and what makes their interaction favorable on a molecular level. This work is helping us better understand how cells repair their damaged DNA in order to prevent persistence of dangerous cancer-causing mutations.
Abstract: Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function.
Pub.: 18 Apr '09, Pinned: 03 Oct '17
Abstract: Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3'-to-5' exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes.
Pub.: 16 Oct '16, Pinned: 03 Oct '17
Abstract: 5-Hydroxymethylcytosine (5hmC) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of 5hmC on the structure, thermodynamics, and conformational dynamics of the Holliday junction (the four-stranded DNA intermediate associated with HR) in its native stacked-X form. The hydroxymethyl and the control methyl substituents are placed in the context of an amphimorphic GxCC trinucleotide core sequence (where xC is C, 5hmC, or the methylated 5mC), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the 5hmC junction adopts two distinct rotational conformations, with an in-base-plane form being dominant over the competing out-of-plane rotamer that has typically been seen in duplex structures. The in-plane rotamer is seen to be stabilized by a more stable intramolecular hydrogen bond to the junction backbone. Stabilizing hydrogen bonds (H-bonds) formed by the hydroxyl substituent in 5hmC or from a bridging water in the 5mC structure provide approximately 1.5–2 kcal/mol per interaction of stability to the junction, which is mostly offset by entropy compensation, thereby leaving the overall stability of the G5hmCC and G5mCC constructs similar to that of the GCC core. Thus, both methyl and hydroxymethyl modifications are accommodated without disrupting the structure or stability of the Holliday junction. Both 5hmC and 5mC are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating 5hmC into a DNA junction are thus discussed in the context of the specificity in protein recognition of the hydroxymethyl substituent through direct and indirect readout mechanisms.
Pub.: 21 Sep '16, Pinned: 03 Oct '17
Abstract: 5-hydroxymethylcytosine (5hmC) has been suggested to be involved in various nucleic acid transactions and cellular processes, including transcriptional regulation, demethylation of 5-methylcytosine and stem cell pluripotency. We have identified an activity that preferentially catalyzes the cleavage of double-stranded 5hmC-modified DNA. Using biochemical methods we purified this activity from mouse liver extracts and demonstrate that the enzyme responsible for the cleavage of 5hmC-modified DNA is Endonuclease G (EndoG). We show that recombinant EndoG preferentially recognizes and cleaves a core sequence when one specific cytosine within that core sequence is hydroxymethylated. Additionally, we provide in vivo evidence that EndoG catalyzes the formation of double-stranded DNA breaks and that this cleavage is dependent upon the core sequence, EndoG and 5hmC. Finally, we demonstrate that the 5hmC modification can promote conservative recombination in an EndoG-dependent manner.
Pub.: 31 Oct '14, Pinned: 03 Oct '17
Join Sparrho today to stay on top of science
Discover, organise and share research that matters to you