Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT) n repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins.

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The observation that transcriptionally active genes generally replicate early in S phase and observations of the interaction between transcription factors and replication proteins support the thesis that promoter elements may have a role in DNA replication. To test the relationship between transcription and replication we constructed HeLa cell lines in which inducible green fluorescent protein (GFP)-encoding genes replaced the proximal ϳ820-bp promoter region of the c-myc gene. Without the presence of an inducer, basal expression occurred from the GFP gene in either orientation and origin activity was restored to the mutant c-myc replicator. In contrast, replication initiation was repressed upon induction of transcription. When basal or induced transcription complexes were slowed by the presence of ␣-amanitin, origin activity depended on the orientation of the transcription unit. To test mechanistically whether basal transcription or transcription factor binding was sufficient for replication rescue by the uninduced GFP genes, a GAL4p binding cassette was used to replace all regulatory sequences within ϳ1,400 bp 5 to the c-myc gene. In these cells, expression of a CREB-GAL4 fusion protein restored replication origin activity. These results suggest that transcription factor binding can enhance replication origin activity and that high levels of expression or the persistence of transcription complexes can repress it.Models for DNA replication in eukaryotes derive significantly from studies of Saccharomyces cerevisiae and Xenopus laevis systems, in which prereplication complexes (pre-RCs) containing the hexameric origin recognition complex ORC, Cdc6, and the minichromosome maintenance proteins are activated by the cell cycle-regulated kinases Cdc7/Dbf4 and cyclin-dependent kinases to unwind DNA and load DNA polymerases for the initiation of DNA synthesis (reviewed in reference 8). Although replication origins in higher organisms do not generally display the conserved size, sequence, or structure of replicators in S. cerevisiae, this overall series of events appears to be conserved in fission yeast and metazoan somatic cells, and the demonstration that chromosomal regions involved in the initiation of replication can promote replication when transferred to ectopic chromosomal sites provides genetic evidence for the existence of defined replicator elements that can control replication initiation in the chromosomes of multicellular organisms (1,2,4,51,52).Replication origins are frequently found upstream of eucaryotic transcription units (reviewed in reference 57), consistent with the suggestion that transcription and replication may be oriented to coordinate transcription and replication fork movement (13,27,50). Transcribed sequences are generally replicated earlier in S phase in the cells where they are expressed than in cells where they are not (22,76), possibly as a result of the modification of chromatin structure by transcription factors (48). However, several investigators report positive (11,19,21,25,34,36,47...