Scott Cheng-Hsin Yang

Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing

Microarrays are powerful tools to probe genome‐wide replication kinetics. The rich data sets that result contain more information than has been extracted by current methods of analysis. In this paper, we present an analytical model that incorporates probabilistic initiation of origins and passive replication. Using the model, we performed least‐squares fits to a set of recently published time course microarray data on Saccharomyces cerevisiae. We extracted the distribution of firing times for each origin and found that the later an origin fires on average, the greater the variation in firing times. To explain this trend, we propose a model where earlier‐firing origins have more initiator complexes loaded and a more accessible chromatin environment. The model demonstrates how initiation can be stochastic and yet occur at defined times during S phase, without an explicit timing program. Furthermore, we hypothesize that the initiators in this model correspond to loaded minichromosome maintenance complexes. This model is the first to suggest a detailed, testable, biochemically plausible mechanism for the regulation of replication timing in eukaryotes.

Reconciling stochastic origin firing with defined replication timing

Eukaryotic chromosomes replicate with defined timing patterns. However, the mechanism that regulates the timing of replication is unknown. In particular, there is an apparent conflict between population experiments, which show defined average replication times, and single-molecule experiments, which show that origins fire stochastically. Here, we provide a simple simulation that demonstrates that stochastic origin firing can produce defined average patterns of replication firing if two criteria are met. The first is that origins must have different relative firing probabilities, with origins that have relatively high firing probability being likely to fire in early S phase and origins with relatively low firing probability being unlikely to fire in early S phase. The second is that the firing probability of all origins must increase during S phase to ensure that origins with relatively low firing probability, which are unlikely to fire in early S phase, become likely to fire in late S phase. In addition, we propose biochemically plausible mechanisms for these criteria and point out how stochastic and defined origin firing can be experimentally distinguished in population experiments.

Replication timing is regulated by the number of MCMs loaded at origins

Replication timing is a crucial aspect of genome regulation that is strongly correlated with chromatin structure, gene expression, DNA repair, and genome evolution. Replication timing is determined by the timing of replication origin firing, which involves activation of MCM helicase complexes loaded at replication origins. Nonetheless, how the timing of such origin firing is regulated remains mysterious. Here, we show that the number of MCMs loaded at origins regulates replication timing. We show for the first time in vivo that multiple MCMs are loaded at origins. Because early origins have more MCMs loaded, they are, on average, more likely to fire early in S phase. Our results provide a mechanistic explanation for the observed heterogeneity in origin firing and help to explain how defined replication timing profiles emerge from stochastic origin firing. These results establish a framework in which further mechanistic studies on replication timing, such as the strong effect of heterochromatin, can be pursued.