Conservation of replication timing reveals global and local regulation of replication origin activity

Abstract

DNA replication initiates from defined locations called replication origins; some origins are highly active, whereas others are dormant and rarely used. Origins also differ in their activation time, resulting in particular genomic regions replicating at characteristic times and in a defined temporal order. Here we report the comparison of genome replication in four budding yeast species: Saccharomyces cerevisiae, S. paradoxus, S. arboricolus, and S. bayanus. First, we find that the locations of active origins are predominantly conserved between species, whereas dormant origins are poorly conserved. Second, we generated genome-wide replication profiles for each of these species and discovered that the temporal order of genome replication is highly conserved. Therefore, active origins are not only conserved in location, but also in activation time. Only a minority of these conserved origins show differences in activation time between these species. To gain insight as to the mechanisms by which origin activation time is regulated we generated replication profiles for a S. cerevisiae/S. bayanus hybrid strain and find that there are both local and global regulators of origin function.

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences

“…Patterns of origin firing at native chromosomes revealed by S-phase read depths (Fig. 1B, in red) coincided with previous results generated using deep sequencing (13,14) and density transfer techniques (18,19). The native chromosomes in this strain completed replication at different times across their length.…”

“…Deep sequencing has been used effectively to dissect the early stages of DNA replication, as read depths change from 1× to 2× over the course of S phase (13,14). Because this transition begins at active replication origins, comparing read depths for S-phase-sorted cells with cells in G1 or G2 has been an efficient and sensitive method to elucidate the starting points for genome duplication.…”

“…We therefore subdivided G2 into early and late fractions and used S-phase cells to identify early origins, as has been done previously (Fig. 1A) (13,14). All read depths were normalized to those in G1.…”

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells

“…This analysis, termed marker frequency analysis (MFA), was designed to study chromosomal properties (Yoshikawa and Sueoka 1963;Altenbern 1971) and was recently applied for capturing genome-wide replication timing (Müller et al 2014). A variant of this method enriches for actively replicating cells by staining the DNA and FACS-sorting S phase population (Schübeler et al 2002;Koren et al 2010;Müller and Nieduszynski 2012;Müller et al 2014). This method does not perturb the cell cycle and requires sequencing a single sample for each mutant.…”

Analysis of Replication Timing Using Synchronized Budding Yeast Cultures

“…Because such a temporal order is readily observed from yeast to humans [2, 3], it is inferred that a well-executed temporal program of replication is crucial for the fidelity of chromosome maintenance and partition. Indeed, altered replication timing has been linked to increased genome instability in yeast [4–6] and, in metazoans, to transcription [7, 8], cell differentiation [4, 9–12], and cancer formation [13–15].…”