5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication

In most mammalian cells, DNA replicationoccurs once, and only once between cell divisions. Replicationinitiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replicationis initiated from a defined consensus sequence, whereas a consensus sequencedelineating mammalian originof replicationhas not been identified. Here we show that 5-hydroxymethylcytosine(5hmC) is present at mammalian replicationorigins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell divisionby increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication- licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.