To elucidate the role of the chromatin environment in the regulation of replication origin activation, autonomously replicating sequences were inserted into identical locations in the budding yeast genome and their activation times in S phase determined.
Early S replicating sequences are assembled into nucleosomes enriched with acetylated histones H3 and H4, the marks of open chromatin, as opposed to late S replicating DNA, which is packaged mainly into silent chromatin marked by deacetylated forms of these same histones [ 107, 108].
Following this functional identification, a linear DNA construct, including the centromere, telomeric DNA from Tetrahymena rDNA termini, and the autonomously replicating sequence, was generated and termed yeast artificial chromosome (YAC) (Murray and Szostak, 1983).
In the third step, de-replicated sequences were sorted by abundance using the information from step 2. In the forth step, sequences were clustered at 97% identity.
The replication origins of the budding yeast S. cerevisiae, also identified as ARS (autonomously replicating sequences), are ∼200 bp (or longer) and contain an essential 11-bp ARS core-A consensus sequence (ACS) [30], [31], extended to a 17-bp motif when based on a larger number of origins [32], [33].
From these studies with higher resolving power than previous cytogenetic analyses, we concluded that while early replicating sequences are found predominantly in G-negative bandsizeableeable portion is not.
The artificially replicated sequences that were an artifact of the 454-based pyrosequencing technique were identified and eliminated using the Replicates software (Gomes-Alvarez et al. 2009).
Verified duplicate files from replicate sequencing were removed from the pool to reduce perceived cluster depth and improve data analysis.
All remaining artifacts (nearly all of which occurred in the reverse direction) that were consistent and reproducible across multiple samples, and with replicate sequencing, were cataloged.
In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition.
In order to generate a consensus sequence the five replicate assemblies, sequences were aligned using kalign [ 23] and a consensus generated using a custom Perl program.
