Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus

Vertebrates have developed multiple mechanisms to coordinate the replicatio n of epigenetic and genetic information. Dnmt1 encodes the maintenance enzy me DNA-methyltransferase, which is responsible for propagating the DNA meth ylation pattern and the epigenetic information that it encodes during repli cation. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequenc e elements associated with previously characterized origins of DNA replicat ion. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardat ion assay, we show that it contains a site for binding of origin-rich seque nces binding activity, a recently purified replication protein. Finally, us ing competitive polymerase chain reaction, we show that replication initiat es in this region in vivo, Based on these lines of evidence, we propose tha t initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.