Determining the contribution of 5-hydroxymethylcytosine to the mutability of DNA

Mutations in the DNA are known to cause or predispose to a number of diseases, resulting in one of the leading origins of non-infectious lethality and long term disability worldwide. For example, it is widely accepted that alterations in the genetic material cause cancer and mounting evidence supports the idea that accumulation of DNA damage is the underlying basis of aging, especially aging related dementia (BBSRC strategic priority: "Ageing research"). Hence, elucidation of the environmental factors and the mechanisms resulting in mutations is critical both for basic understanding of DNA function and developing preventative strategies for decreasing of the mutational load on DNA.
DNA is composed of four bases. Due to a different chemical nature, each of the four bases has a unique propensity to respond to a damaging agent and exclusive repair enzymes to repair it. We focus on one of the four bases in the genome - cytosine, which is a distinctive base in the sense that it is the only base known to receive biologically important modifications in vertebrates. We aim to extend the knowledge of how biological modifications of the cytosine base affect information stability in the DNA.
The precedent that the rate of mutations can be affected by a biological modification of cytosine to 5-methylcytosine (5mC) is well established. For example, current sequencing efforts have demonstrated that 5mC containing sequences are the mutational hot spots in cancer and other genetic disorders.
We have recently identified a novel DNA modification, 5-hydroxymethylcytosine (5hmC) that is detectable in all normal cell types and particularly enriched in terminally differentiated tissues such us brain and kidney. The mutagenic potential of 5hmC is not known. Interpretation of our preliminary data is compatible with the hypothesis that increased abundance of 5hmC in the DNA of brain cells serves to protect neuronal cells from increased DNA damage. Since neuronal cells are irreplaceable without the consequence of information loss, these cells have evolved additional means to protect their DNA, and the conversion of 5mC to might be an important mechanism in guarding the integrity of the genome.
We will employ a range of state-of-the-art techniques to determine the mutation rates of 5hmC in cells and in mice, and to identify the biological pathways which influence DNA mutagenesis and repair. The results of this proposed research will lay a foundation and inform any future translational studies aiming to modulate DNA modifications.

It has been widely recognized that CpGs in the genome are mutational hot-spots both in germline and somatic tissues. Over 30% of human disease causing mutations arise in CpG dinucleotides. The mutational load on CpG dinucleotides is partly explained by the fact that CpGs in the genome are the primary substrate of methyltransferases, which generate 5-methylcytosine (5mC). Two fold higher deamination rate and less efficient base excision repair of T-G mismatch resulting after deamination of 5mC have been attributed as the main cause of increased mutation rate in CpGs. Reflection of 5mC mutability is seen in our genomes, where nearly fivefold lower genome-wide CpG frequency is observed to the randomly expected one.
We have recently identified 5-hydroxymethylcytosine (5hmC) in the genome and showed that in neuronal cells it is found along the whole transcribed region of highly expressed genes. What the mutagenic potential of 5hmC is in relation to unmodified or methylated counterpart is not known. We propose evaluating in vitro and in vivo mutagenic potential for 5hmC and the environmental (UV) and cell intrinsic determinants (deaminases, glycosylases) of its mutability. The outcomes will elucidate the novel genome maintenance strategies, which were evolved to achieve the fine balance between information fidelity leading to stable perpetuation of living organisms and infidelity, which is a driving force of evolution.

The proposal is designed to expose the fundamental property of information stability in the DNA by elucidating how 5-hydroxymethylcytosine (5hmC) affects cytosine mutation rate in the genome. As indicated above (see academic beneficiaries) the results are expected to be of interest to a wide group of academics in the fields of epigenetics, DNA damage-repair, cancer, neuroscience and ageing. By examining the fundamental biological question we will provide groundwork for directly clinically relevant projects bringing both academics and pharmaceutical industry to exploit how activity of DNA modifying enzymes can be regulated to promote information stability for prevention of diseases caused by alterations in DNA. These longer term outcomes have the potential to benefit the public health by preventative strategies utilising the biochemical mechanisms of mutations in the DNA.
We increase impact of our research through outreach activities. Our outreach activities target both general society and specific groups. We are delegating time and effort to educate and motivate school pupils by providing a work experience program. During a two week period they work together with researchers, gaining both science education and insights into scienctific career opportunities. General society is reached by active participation in science festivals and CRUK organised events. Additionally, the Ludwig Institute for Cancer Research, University of Oxford has a Scientific Communications Manager to ensure that our work is widely publicised and co-ordinates outreach activities with other departments within the University of Oxford to obtain maximum impact.