The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

A novel cloning strategy for sequences comprising mammalian replication origins, described by Mesner et al. (2006) in a recent issue of Molecular Cell, utilizes an origin trapping assay in which replication bubbles are selectively retained in agarose due to their circular nature.

Advances in microarray technology have enabled the analysis of replication dynamics on a genome-wide scale, providing deeper insight to the factors that regulate DNA replication. Studies using high-density microarrays have led to the genome-wide identification of replication origins in the budding yeast, Saccharomyces cerevisiae, and enabled the analysis of the global temporal pattern of origin activation under various conditions. We have developed a replication origin array that contains the approximately 430 potential origins in the yeast genome. By detecting the copy number change that occurs as cells progress from G1 to S phase on these arrays, we have produced origin activation patterns in wild-type cells similar to those obtained from previous studies that used whole-genome arrays. We have also applied this method to study S phase checkpoint mutants, providing insight into the genome-wide regulation of replication origin activation by S phase checkpoint kinases in the presence of replication stress. The main procedures of this technique involve arresting yeast cells in G1 and S phase, isolating and labeling genomic DNA with fluorescent dyes, and cohybridizing the DNA samples to replication origin arrays to yield copy number change data.