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Cis-Acting Elements
Initiation of DNA replication in Escherichia coli, mammalian viruses, and the budding yeast Saccharomyces cerevisiae is controlled primarily by trans-acting initiator proteins that interact with cis-acting DNA sequence elements (the replicators). In these simple replicons, the cis-acting element consists of an essential core sequence, containing initiator protein binding sites and easily unwound sequences (DNA unwinding elements [DUEs]), and auxiliary sequences which enhance the efficiency of replication initiation. In S. cerevisiae, the initiation of replication requires an autonomously replicating sequence (ARS) element in cis and occurs within a region flanked by a DUE and the binding sites for the origin recognition complex (ORC) and other initiator proteins. The initiation site for leading-strand synthesis appears to be restricted to a single nucleotide in a chromosomal ARS element. Replication initiation in fission yeast is also controlled by the sequence-specific recognition of a cis-acting replicator by ORC and associated initiator proteins, but the fission yeast replicators are larger than those of its budding yeast counterpart. Initiation activity of yeast ARS elements in chromosomal DNA depends not only on specific DNA sequences in the ARS but also on chromatin structure and chromosomal position.
Extensive mapping of replication start sites in mammalian chromosomes has revealed that replication at most but not all loci begins at a few high-frequency start sites contained within a broad zone of initiation. One of the most thoroughly mapped high-frequency initiation regions (IRs) in mammalian chromosomes is the region downstream from the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. This region contains a 55-kb zone of delocalized origin activity containing three preferred start sites: origin beta (ori-), centered approximately 17 kb downstream from the DHFR gene; ori-' just downstream from ori-; and ori-, located 23 kb further downstream.
Sequences at a Mammalian Origin
Altman AL, Fanning E., (2001) The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity. Mol Cell Biol. 2001 Feb;21(4):1098-110.
A 5.8-kb fragment of the DHFR ori- region placed in random ectopic chromosomal locations is sufficient to direct initiation of DNA replication from the ori- start site, and functions as an independent chromosomal replicator. In the ectopic DHFR ori- region, at least two well-separated DNA sequence elements were critical for full activity. Deletion of the GA dinucleotide repeat in pMCDDNR or the central AT-rich sequence in pMCDAT reduced initiation activity nearly 10-fold. Similarly, GA dinucleotide repeats direct the establishment of a functionally important nucleosomal array in the transcription control region upstream of a Drosophila heat shock gene and could play such a role in the ectopic DHFR ori- region. The central AT-rich region that was deleted in pMCDAT harbors a potential DUE (16) and sequences homologous to a cell cycle-dependent protein footprint in the human lamin B2 and to the ORC binding sites in ACE3. Either DNA unwinding or protein binding could account for the requirement for the central AT-rich region in the 5.8-kb ori- fragment. DNA sequences in the 5.8-kb DHFR fragment are sufficient to direct efficient initiation at the ori- IR in multiple ectopic chromosomal sites and that initiation activity depends on discrete genetic elements located at or near the IR. Thus, our lab is working on further characterizing these elements which is required to confirm their proposed modular nature and to elucidate their biochemical functions.
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