Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Location, location, location: it’s all in the timing for replication origins Oscar M. Aparicio1 Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089, USA The differential replication timing of eukaryotic replica- enabled the first detailed studies of chromosomal DNA tion origins has long been linked with epigenetic regula- replication origins in eukaryotic cells (Stinchcomb et al. tion of gene expression and more recently with genome 1979). ARSs were identified as relatively short (100- to stability and mutation rates; however, the mechanism has 200-base-pair [bp]) chromosomal DNA sequences that remained obscure. Recent studies have shed new light by confer replication to plasmids, allowing their mitotic identifying novel factors that determine origin timing in propagation (for review, see Newlon and Theis 1993). yeasts and mammalian cells and implicate the spatial Further analysis of these sequences within their native organization of origins within nuclear territories in the chromosomal loci confirmed their function as replication mechanism. These new insights, along with recent find- origins within chromosomes. As prototypes for replica- ings that several initiation factors are limiting relative to tion origins in higher cells, ARS studies in yeast propelled licensed origins, support and shape an emerging model for much of the genetic and biochemical elucidation of the replication timing control. The mechanisms that control macromolecular events involved in replication initiation the spatial organization of replication origins have poten- in all eukaryotes. Although yeast origins differ in se- tial impacts for genome regulation beyond replication. quence specificity from mammalian origins, both share highly conserved regulation at the molecular and cellular levels (for review, see Cvetic and Walter 2005). Eukaryotic replication origins are governed by a series of protein recruitments, beginning with binding of the Scope origin recognition complex (ORC), which marks poten- Several excellent reviews have recently examined the tial sites of origin assembly (for review, see Bell and Dutta significance of replication timing in epigenetic gene 2002). In G1 phase, ORC, Cdc6, and Cdt1 together load regulation and reprogramming, genetic instability, and inactive minichromosome maintenance (MCM) helicase mutation rates and evolution (for review, see Aladjem complexes at origins. This MCM loading step, which is 2007; Gondor and Ohlsson 2009; Mendez 2009; Gilbert also referred to as ‘‘licensing’’ or ‘‘pre-RC assembly,’’ et al. 2010; Natsume and Tanaka 2010; Burgess 2011; potentiates origins for initiation. Initiation is controlled Herrick 2011). Despite substantial progress in character- by Dbf4-dependent kinase (DDK) and cyclin-dependent izing replication timing in various model systems, our kinase (CDK), which are sequentially activated during the understanding of the underlying mechanisms that estab- G1-to-S-phase transition (for review, see Labib 2010). lish the timing of origin activation has remained rather DDK acts first, phosphorylating MCM subunits to load vague. This review highlights recently published studies Cdc45 and Sld3 (Heller et al. 2011). Next, CDK phos- that provide new mechanistic insights by identifying phorylates Sld3 and Sld2, leading to the recruitment of novel factors that control replication timing throughout GINS (which, together with Cdc45, stimulates MCM the budding yeast and fission yeast genomes. These helicase) and additional factors, including DNA poly- factors are conserved in higher eukaryotes, and one has merases, to assemble a complete replisome and initiate already been shown to control replication timing in DNA synthesis. Whereas this requisite sequence of mammalian genomes, where replication timing is linked events is shared by all active origins, they differ in timing with gene regulation and the developmental program. and efficiency of initiation during S phase. Timing and efficiency are distinct measures that are Replication origins and initiation control frequently used to characterize origin activity. These measures are population averages and are typically de- The discovery of autonomously replicating sequences termined through distinct methods (Kalejta and Hamlin (ARSs) in the budding yeast Saccharomyces cerevisiae 1996; Huberman 1997; van Brabant et al. 1998). Because origins differ in their characteristic initiation timing and [Keywords: replication timing; chromatin; Fkh1; Fkh2; Rif1; Taz1] exhibit substantial variance in this timing across a pop- 1Correspondence E-mail [email protected] ulation, replication forks from adjacent origins some- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.209999.112. times replicate origins ‘‘passively,’’ precluding initiation. GENES & DEVELOPMENT 27:117–128 Ó 2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; www.genesdev.org 117 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Aparicio Thus, most replication timing measurements represent Due to differences in origin efficiencies and timings, a composite of initiation time and passive replication different subsets of origins are used in different cells of time. Efficiency is the resulting frequency of initiation a population (Fangman and Brewer 1991; Kalejta and (Friedman and Brewer 1995; Kalejta and Hamlin 1996). Hamlin 1996; Patel et al. 2006; Czajkowsky et al. 2008). The replication timing and efficiency of neighboring Nevertheless, at the population level, replication timing origins are interrelated in that earlier or more efficient profiles emerge reflecting individual origins’ timings and origins will often passively replicate their later or less efficiencies. Complete analysis of origin timings and efficient neighbors. Consequently, timing and efficiency efficiencies of all known origins on S. cerevisiae chromo- are correlated with earlier origins generally being more somes III and VI, confirmed by whole-genome studies, has efficient and later origins being less efficient (Yamashita revealed general features of chromosomal replication, et al. 1997; Yang et al. 2010). Both timing and efficiency including early replication of at least one origin near each may be thought of as reflecting the probability of origin CEN, late firing and/or dormancy of origins in subtelo- firing, with early timing reflecting a high probability per meric regions as well as those associated with the hetero- unit time (at least in early S phase) and high efficiency chromatic silencers, and a wide range of origin timings and reflecting a high, cumulative probability over the entire efficiencies throughout other chromosomal regions, with period of S phase. It has been suggested, therefore, that some origins notably earlier than the CEN-proximal ori- timing and efficiency derive from a common underlying gins (Newlon et al. 1993; Friedman et al. 1997; Yamashita mechanism that determines these probabilities (Rhind et al. 1997; Poloumienko et al. 2001; Raghuraman et al. 2006). The central challenge has been to define this 2001; Yabuki et al. 2002). Together with the ARS re- mechanism. location studies, these findings implicated some aspect of these different chromosomal domains, such as chromatin structure, transcription, or subnuclear localization, in Chromosomal position and chromatin structure origin control. Chromosomal context and local chromatin structure are The idea that chromatin structure, subnuclear locali- major determinants of replication origin timing and zation, and/or gene expression regulate initiation gained efficiency, as demonstrated by seminal ARS relocation direct support from studies that analyzed replication in experiments (Ferguson and Fangman 1992). This study strains with deletions of chromatin modifiers and related found that ARS501 residing near a subtelomeric region factors, such as SIR3, which is required for assembly of initiated replication later than ARS1 residing near a cen- subtelomeric heterochromatin; YKU70, which is re- tromere (CEN). Both origins initiated efficiently, indicat- quired for telomere localization to the nuclear periphery; ing that both were efficiently licensed and competent to and RPD3, which deacetylates histones to regulate gene fire. Relocating ARS1 to a subtelomeric location near expression. Deletion of SIR3 resulted in earlier replica- ARS501 delayed its replication, whereas ARS501 initi- tion of subtelomeric origins, and when early origin ARS1 ated early when relocated outside of the subtelomeric was transplanted into a subtelomeric region, its timing region (onto a plasmid). These findings suggested that was delayed in a SIR3-dependent manner (Stevenson and early firing is the normal state and that late timing is Gottschling 1999). Deletion of YKU70 also advanced imposed by repressive chromatin flanking the origin, replication timing of subtelomeric origins, suggesting such as subtelomeric heterochromatin. Supporting and a role for localization of subtelomeric chromatin to the extending this idea, late-firing origins were identified in nuclear periphery (Cosgrove et al. 2002). Moreover, de- nontelomeric regions that fired early when located on letion of RPD3 advanced the timing of most nontelo- a plasmid but fired later when 6–15 kb of native, flanking meric, late-firing origins, and tethering GCN5 histone