SnapShot: Replication Timing Benjamin D. Pope,1 Oscar M. Aparicio,2 and David M. Gilbert,1 1Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA 2Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA YEAST MAMMALS Replication Replication foci foci 5 µm REPLICATION DOMAINS NUCLEAR INTERIOR 1 µm Early replicating x x x Differentiation x Replication NUCLEAR LAMINA Late replicating Constitutively early Developmentally regulated Constitutively late GC rich Intermediate sequence composition AT rich Gene rich Low nuclease sensitivity Gene poor Prereplicative complex High nuclease sensitivity Dynamic chromatin marks Low nuclease sensitivity Replisomes Correlated with transcription Unreplicated DNA Replicated DNA Interdomain interaction cis boundary S-phase Replication Number of Number of Number of Replicons/ Number of Species Genome size length fork rate potential origins replicons per S foci per S focus domains Domain size Yeast 12-14 Mb <1 hr 1-2 kb/min 500-1,000 100-200 15-30 4-7 40-70? ≤250 kb Mammals 2-4 Gb 8-10 hr 1-2 kb/min >250,000 25,000-50,000 5,000-10,000 6-20 4,000-5,000 400-800 kb MAMMALS High chromatin Chromosome mobility Domains repositioned at the TDP remain anchored for the remainder of interphase condensation Pre-RC Timing decision Origin decision Restriction Early Late Timing assembly point (TDP) point (ODP) point replication replication determinants lost M G1 EARLY S LATE S G2 M YEAST Timing decision point (TDP) Early replication Late replication Prereplicative complex Early origin Cdc45 Limited pool Fkh1/2 Rif1, Taz1, Yku70 Late origin or Rpd3 Cdc45 loading stimulated by Initiation of Cdc45 loaded Replisome Fkh1/2 and inhibited by origins and removal of Terminating replisomes Rif1, Taz1, Yku70 or Rif1/Taz1/Yku70/Rpd3- release Cdc45 to activate Rpd3 deacetylation mediated repression unred origins 1390 Cell 152, March 14, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.02.038 See online version for legend and references. SnapShot: Replication Timing Benjamin D. Pope,1 Oscar M. Aparicio,2 and David M. Gilbert,1 1Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA 2Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA All organisms use similar principles to duplicate DNA at replication forks (Yao and O’Donnell, 2010). However, eukaryotic cells contain large chromosomes with hundreds to thousands of replication origins and complex, heterogeneous chromatin. Conserved cell-cycle and checkpoint mechanisms ensure one complete round of replication (Labib, 2010), and additional mechanisms coordinate initiation at the many replicons (regions replicated from a single origin) in space and time. A temporal order to genome replication balances replication with limiting cellular resources such as initiation factors and nucleotide pools (Aparicio, 2013; Rhind and Gil- bert, 2013). Chromatin features regulate replication timing by controlling the access of initiation factors to replication origins. In fact, replication timing is one of the few cellular functions that are clearly regulated at the level of large-scale/long-range chromatin folding. In mammals, this temporal order is regulated during development and is linked to transcriptional regulation (Nordman and Orr-Weaver, 2012; Rhind and Gilbert, 2013). Replication foci in budding and fission yeast are nuclear sites of active replication that can be visualized by fluorescently tagged replication fork proteins or nucleotide ana- logs (Kitamura et al., 2006; Meister et al., 2007). Foci (yellow) in the displayed image (originally published in Trends Cell Biol., December 2001) exhibit a pattern that is typical of early S in budding yeast. Chromatin is counterstained (red). These foci are mobile and frequently fuse with other foci or split to form new foci, making precise measurements of their characteristics difficult. The ratio of replication foci to active replication forks indicates that several closely spaced replicons are active simultaneously within each focus, although the organization can only be modeled imprecisely at this point. Replication foci in mammals are more numerous than in yeast and, relative to the size of the nucleus, less mobile (Maya-Mendoza et al., 2010; Rhind and Gilbert, 2013). An average focus replicates ~1 Mb of DNA in 45–60 min. In the displayed image (originally published in Genome Res., June 2010), cells were dual labeled in successive pulses to visualize both early (green) and late (red) foci simultaneously, highlighting the spatial compartmentalization of chromatin replicated at different times during S phase. Foci labeled in one cell cycle are stable in appearance for many cycles, indicating that they are structural units and most likely the cytological equivalents of replication domains measured by molecular genomics methods. Like foci, replication domains are chromosomal units that are replicated coordinately by synchronously firing clusters of replicons, which also approach megabase size in mammals. At least half of replication domains are regulated to replicate at different times in different tissues (Nordman and Orr-Weaver, 2012; Rhind and Gilbert, 2013). Replicons associated with the nuclear interior are replicated early in S phase, whereas those adjacent to the lamina replicate later. Domains that switch replication timing during differentiation move between subnuclear compartments, as indicated both by physical position and by changes in interdomain chromatin interactions (Takebayashi et al., 2012). Replication domain boundaries may insulate chromatin types from each other, facilitating the differential replication timing of adjacent chromatin domains. In yeast, the extent to which clusters of origins form replication domains is controversial. At least four large (~250 kb) regions in budding yeast have distinctly late timing (McCune et al., 2008). In addition, each chromosome could be considered to contain several domains—the centromere, each arm, and the telomeres—possibly with a few addi- tional subdomains. The range in number of potential origins reflects differences in budding (~500) and fission yeast (~1,000), wherein origin efficiencies are generally higher in budding yeast, probably resulting in similar replicon numbers. Exiting mitosis, the chromatin of mammalian cells is highly mobile and lacks determinants for replication timing. Within 1–2 hr, cells reach the timing decision point (TDP), when replication domains/foci anchor in their respective subnuclear positions for the remainder of interphase and simultaneously acquire the ability to dictate a replication-timing program (Rhind and Gilbert, 2013). Establishing this timing program occurs upstream of specifying which sites will be used for initiation (origin decision point) and the activation of S phase Cdk activity (restriction point). The replication-timing program is executed during S phase through the firing of several sequential groups of internally localized foci (green), followed by replication at the nuclear and nucleolar periphery (red) and, finally, a few sites of internally localized heterochromatin (not shown). Chromatin in G2 phase lacks determinants for replication timing, suggesting that such determinants are lost during replication. Coincident with the TDP (between mitosis and start in yeast), Fkh1/2 and Rif1/Taz1/Yku70/Rpd3 organize early and late-replicating chromatin, respectively. Fkh1/2 bind con- sensus DNA elements near early replicating origins and through interaction with the origin recognition complex (ORC) and/or through Fkh1/2 dimerization bring the origins into proximity (Aparicio, 2013). Fkh1/2 stimulates Cdc45 recruitment and loading onto prereplication complexes (pre-RCs) at early origins during G1 phase, facilitating early initiation. Rif1/Taz1/Yku70 position and tether telomeres and some internal sequences to the nuclear periphery, whereas Rpd3 modifies chromatin, isolating late-replicating chromatin from Cdc45 (and other initiation factors). 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