Genome-wide Mapping of DNA Synthesis in Saccharomyces cerevisiae Reveals That Mechanisms Preventing Reinitiation of DNA Replication Are Not Redundant

Abstract

To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.

A ‘DNA replication’ signature of progression and negative outcome in colorectal cancer

“…genes contributes to colorectal tumorigenesis. This could be linked to an increase in the number of active origins of replication (and therefore re-replication and aneuploidy), as already shown in yeast (Green et al, 2006).…”

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

“…[13][14][15][16] The deregulation of any one pre-RC component is not sufficient to induce DNA re-replication. [14][15][16][17] The simultaneous deregulation of two components induces limited origin re-firing in a subset of origins, while deregulation of ORC, Cdc6, and the MCM2-7 complex together induces more substantial DNA re-replication. 14,16,17 The simultaneous deregulation of Cdt1 with the other three components has not been reported.…”

Cyclin and Cyclin-Dependent Kinase Substrate Requirements for Preventing Rereplication Reveal the Need for Concomitant Activation and Inhibition

“…org/supplemental/). Recently, Green et al (2006) showed little or no replication initiation defects associated with ORC, MCM, and CDC6 mutations deregulating replication control by a microarray-based assay. Thus, overall we consider it unlikely that replication initiation defects contribute significantly to the results presented here.…”

“…For these reasons, we consider that this DNA flow cytometry signal likely represents authentic rereplication, but we recognize that in borderline cases it is hard to be sure of this. Recently, Green et al (2006), using a microarray method, showed that rereplication at some specific loci could be detected even in cells without DNA flowcytometry-detectable rereplication, and such an assay may ultimately be necessary to definitively prove rereplication in cases that are borderline by DNA flow cytometry.…”