The Consequences of DNA Re-replication
- Author(s): Green, Brian Michael
- Advisor(s): Li, Joachim J
- et al.
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. Joachim's lab had previously shown 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. I have helped to refine this understanding and have identified a number of deleterious consequences of re-replication. First, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of rereplication. 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 also showed 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.
I was next interested in both the short and long term consequences of re-replication. I first focused on short term consequences and demonstrated that re-replication rapidly blocks cell proliferation and activates the classical DNA damage-induced checkpoint response. Strikingly, re-replicating cells accumulate subchromosomal DNA breakage products, demonstrating that re-replication leads to DNA double strand breaks.
To address the long term consequences of re-replication, I decided to focus on gene amplification. Gene amplification, a stable increase in the copy number of a region of DNA, is frequently observed in tumors and is thought to be a driving force in tumorigenesis. There are numerous cases of tumor cells, however, with amplicons that are not readily explained by current models. I demonstrated that re-replication is a potent inducer of gene amplification that generates structures similar to these previously unexplained amplicons. The high frequency at which these amplification structures are generated is specific to re-replication, as similar structures are not observed when S phase DNA replication is impaired or DNA is directly damaged. I thus propose that re-replication arising from loss of replication control is a potential source of the genomic instability important for tumorigenesis.