The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is ϳ60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G 1 to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.The initiation of DNA replication in eukaryotes relies on the sequential assembly of protein complexes at replicator sequences, controlled by the activities of kinases and phosphatases (1). Genetic and biochemical studies in Saccharomyces cerevisiae, Drosophila melanogaster, and Xenopus laevis suggest that the origin recognition complex (ORC) 1 is a component of the replication initiator that recruits Cdc6, Cdt1, and the minichromosome maintenance (MCM) proteins to origins late in mitosis to form the pre-replication complex (pre-RC). Activation of the pre-RC for replication requires the activity of S phase cyclin-dependent kinases plus the Cdc7/Dbf4 kinase and involves binding of MCM10, Cdc45, and replication protein A (RPA) to unwind DNA and to load DNA polymerases. A complex containing MCM proteins and Cdc45 may function as a replicative helicase to extend the unwound origin DNA (2, 3).In S. cerevisiae, chromosomal replication origins cloned in plasmids display autonomously replicating sequence (ARS) activity and characteristically comprise a set of modular elements, including an ARS consensus sequence (ACS) (4)-binding site for ORC (5), a region of helical instability termed a DNAunwinding element (DUE) that contributes to origin activity through template unwinding or binding of pre-RC proteins (6 -10), and transcription factor-binding sites that can promote the assemb...
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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.