Abstract
Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. 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 retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.
Highlights
Mammalian DNA replication initiates from multiple sites throughout S phase [1]
The ability of certain specific sequences to support autonomous replication of plasmids has been directly validated by mapping of the same sequences as chromosomal initiation sites for replication in living cells [16]
Using several lines of evidence based on autonomous replication assays, origin binding activity (OBA) binding assays and in vivo mapping by competitive PCR, we propose that the 5Ј region of the dnmt1 gene comprises functional initiation sites for DNA replication
Summary
Mammalian DNA replication initiates from multiple sites throughout S phase [1]. These sites are determined both by cis-acting DNA sequences, known as replicators, and by transacting elements, defined by initiator proteins that bind to the replicator [1, 2]. A number of techniques, including methods for the isolation of newly synthesized DNA in combination with competitive PCR techniques, have led to the identification of new mammalian replication initiation regions as well as characterization of additional initiation sites at established origins [3,4,5,6,7,8]. The ability of certain specific sequences to support autonomous replication of plasmids has been directly validated by mapping of the same sequences as chromosomal initiation sites for replication in living cells [16]. Because of the limited number of mammalian origins of replication that have been characterized far, it is still too early to draw general conclusions regarding the critical genetic and epigenetic determinants of origin function and its differential regulation. Additional origins have to be characterized to allow for understanding of the general rules governing origin function in mammals
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
