The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

Abstract

Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G 1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G 1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.In their replicon model (29), Jacob et al. proposed that replication initiation involves the recognition of a cis-acting DNA sequence or replicator by a trans-acting positive regulatory factor called the initiator. Since then, this model has been validated in bacteria and virus systems, but in eukaryotes a more complex model has been proposed to explain the diversity of replicator structures (22,66). With the exception of budding yeast, a specific sequence element that defines the replication origin in eukaryotes has not been identified (2,6,7,21,23). Instead, it appears that a complex combination of primary DNA sequence and epigenetic factors dictates where replication initiates, and a unique combination of these factors may define the position of each initiation site (22). Although replication in most eukaryotic organisms does not initiate at random with respect to DNA sequence, the size and distribution of these sites are highly variable (for a review, see reference 23). In gene-dense regions, replication can initiate at very defined sites (1, 5), while in large intergenic regions multiple initiation sites are found throughout large (5-to 50-kb) initiation zones (11,13,28,39,45,50).A large (50-kb) region downstream of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells is the most extensively studied initiation zone, yet the precise distribution of initiation sites within this region has still not been resolved. High-resolution mapping of the locations of small nascent DNA strands within a 12-kb region detected two very specific initiation sit...

MYC and the Control of DNA Replication

“…Given the unparalleled extent of MYC binding across the genome Orian et al 2003;Lin et al 2012;Nie et al 2012) and our limited knowledge on how origins of replication are selected in every cell cycle to initiate productive DNA synthesis, the implications for such studies would be far-reaching. The close relationship between MYC chromatin binding and global transcription could provide important clues to understand how the long appreciated connection between transcription and replication origin selection is established (MacAlpine et al 2004;Kohzaki and Murakami 2005;Sasaki et al 2006;Sequeira-Mendes et al 2009;Karnani et al 2010;Dellino et al 2013). Moreover, these studies would also help us understand how MYC influences cell differentiation and facilitates cellular reprogramming (Wilson et al 2004;Cartwright et al 2005;Leon et al 2009;Smith et al 2010), because it is plausible that changes in the distribution, choice, and timing of active replication origins throughout the genome would underlie cell fate transitions (Gilbert 2010;Ryba et al 2011).…”

Heterochromatin protein 1 (HP1) modulates replication timing of the Drosophila genome

“…It seems plausible that transcription could locally interfere with proper origin activation and delay replication timing as a consequence. Indeed, transcription can have a negative influence on origin selection in mammalian cells (Mesner and Hamlin 2005;Gregoire et al 2006;Sasaki et al 2006). It has been suggested that eukaryotic replication origins preferentially locate in intergenic, nontranscribed regions, yet in proximity to actively transcribed genes (MacAlpine and Bell 2005;Macalpine et al 2010).…”

Programming DNA replication origins and chromosome organization

“…In S. pombe as well as in higher eukaryotes, DNA replication origins are often located at promoters (Cadoret et al 2008;Dai et al 2005;Sequeira-Mendes et al 2009; our unpublished data). Transcription factors can affect DNA replication origin localization or activation in different systems (Cheng et al 1992;Danis et al 2004;Ghosh et al 2004;Maric et al 2003;Minami et al 2006;Sasaki et al 2006). This could be achieved by recruiting chromatin remodeling and histone-modifying complexes (Fig.…”