The Involvement of 5-Hydroxymethylcytosine in Two DNA Metabolism Pathways: Transcription and double stranded DNA Breaks

5-hydroxymethylcytosine (5hmC) is involved in various cellular processes, including transcriptional regulation, demethylation of 5-methylcytosine (5meC) and stem cell pluripotency. 5hmC is formed by oxidation of 5-methylcytosine by the Tet family of enzymes. This thesis describes two functions attributed 5hmC – these are the role of 5hmC in transcription and the role of 5hmC in hepatocyte polyploidy. Overwhelming evidence supports the notion that 5meC has a negative effect on transcription; however, only recently has the effect that 5hmC has on transcription begun to be studied. Using model substrates including the CMVIE promoter and a generic gene body we have directly assessed the effect that 5hmC, both at the promoter and in the gene body, has on in vitro gene transcription. We show that the presence of 5hmC modifications strongly represses transcription. We also demonstrate that the inhibition of transcriptional activity is primarily due to the presence of 5hmC in the promoter and that 5hmC in the gene body has a minimal effect on transcription. Thus, we propose that the presence of 5hmC in promoter prevents the binding of essential transcription factors or recruits factors that repress transcription. In the second part of my thesis, we describe the identification of an activity that preferentially cleaves 5hmC-modified DNA; a mammalian nuclease that can differentiate between cytosine, 5meC, and 5hmC. Using biochemical methods this activity was isolated from mouse liver extracts and the enzyme responsible for the cleavage of 5hmC-modified DNA was identified to be Endonuclease G (EndoG). Recombinant EndoG preferentially recognizes and cleaves a core sequence when one specific cytosine within that core sequence is hydroxymethylated. EndoG catalyzes the formation of double stranded DNA breaks in a 5hmC-dependent manner in vitroand in vivo. Finally we show that EndoG and 5hmC can initiate recombination in vitro