The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

Origin Replication Complex Binding, Nucleosome Depletion Patterns, and a Primary Sequence Motif Can Predict Origins of Replication in a Genome with Epigenetic Centromeres

ABSTRACTOrigins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeastCandida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns inSaccharomyces cerevisiaeandKluyveromyces lactisto train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in theC. albicansgenome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A “mini-ARS screen” identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion.IMPORTANCEDNA replication machinery is highly conserved, yet the definition of exactly what specifies a replication origin differs in different species. Here, we utilized computational genomics to predict origin locations inCandida albicansby combining locations of binding sites for the conserved origin replication complex, necessary for replication initiation, together with chromatin organization patterns. We identified predicted sequences that exhibited bona fide origin function and developed a linear plasmid assay to delimit the DNA fragments necessary for origin function. Additionally, we found that a short AC-rich motif, which is enriched in predicted origins, is required for origin function. Thus, we demonstrated a new machine learning paradigm for identification of potential origins from a genome with no prior information. Furthermore, this work suggests thatC. albicanshas two different types of origins: “hard-wired” arm origins that rely upon specific sequence motifs and “epigenetic” centromeric origins that are recruited to kinetochores in a sequence-independent manner.