Loss of Rereplication Control inSaccharomyces cerevisiaeResults in Extensive DNA Damage

Abstract

To maintain genome stability, the entire genome of a eukaryotic cell must be replicated once and only once per cell cycle. In many organisms, multiple overlapping mechanisms block rereplication, but the consequences of deregulating these mechanisms are poorly understood. Here, we show that disrupting these controls in the budding yeast Saccharomyces cerevisiae rapidly blocks cell proliferation. Rereplicating cells activate the classical DNA damage-induced checkpoint response, which depends on the BRCA1 C-terminus checkpoint protein Rad9. In contrast, Mrc1, a checkpoint protein required for recognition of replication stress, does not play a role in the response to rereplication. Strikingly, rereplicating cells accumulate subchromosomal DNA breakage products. These rapid and severe consequences suggest that even limited and sporadic rereplication could threaten the genome with significant damage. Hence, even subtle disruptions in the cell cycle regulation of DNA replication may predispose cells to the genomic instability associated with tumorigenesis. INTRODUCTIONEukaryotic DNA replication is tightly controlled such that every segment of the genome is replicated once and only once each cell cycle. This control is primarily exerted at the hundreds to thousands of replication origins where DNA replication initiates. Once an origin initiates in S phase, multiple mechanisms prevent it from reinitiating replication for the remainder of that cell cycle (Gopalakrishnan et al., 2001;Nguyen et al., 2001;Vas et al., 2001;Yanow et al., 2001;Vaziri et al., 2003). Such tight control suggests that even an occasional reinitiation event would be deleterious to cells, and it is readily apparent that, in principle, excessive synthesis of just small segments of the genome could eventually threaten its stable propagation. Nonetheless, a direct analysis of the consequences of rereplication is needed to understand whether and how rereplication contributes to genomic instability. S. cerevisiae provides a powerful genetic system for such an analysis, especially as there is considerable understanding of both the mechanisms regulating replication and those protecting genome stability in this organism.Eukaryotic replication initiation can be divided into two fundamental stages (reviewed in Bell and Dutta, 2002). In the first stage, which occurs in early G1 phase, a prereplicative complex (pre-RC) is assembled at replication origins through the sequential loading of the initiation proteins origin recognition complex (ORC), Cdc6, Cdt1, and Mcm2-7. In the second stage, activation of two kinases, Dbf4-Cdc7 kinase and a cyclin-dependent kinase (CDK), triggers events that culminate in replication initiation and disassembly of the prereplicative complex: additional replication proteins are recruited to the origin, the DNA is unwound, and replisomes are assembled at two nascent replication forks.In addition to triggering initiation, CDKs also prevent reinitiation of eukaryotic DNA replication (Broek et al., 1991;Dahmann et al., 1995;Sauer et al., 1...

Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage

“…The incorporation of EdU into distinct foci was seen in 9% of atxr5,6 nuclei, but was rarely observed in wild type (<0.1%, Fig regions [i.e., being more abundant in pericentromeric heterochromatin than in the arms of the chromosomes (12)], it should be noted that processes other than overreplication, such as replication-dependent DNA repair, could also account for or contribute to this pattern of EdU incorporation. Overreplication is known to lead to genomic instability and DNA damage, including double-strand breaks (24)(25)(26)(27)(28). Consistent with this observation, many DNA damage-response genes, including BRCA1, PARP1, CYCB, and RAD51 (Fig.…”

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

“…First, if replication controls are highly redundant, the probability that a cell will spontaneously acquire the multiple disruptions needed to induce re-replicate will be extremely small. Second, we and others have shown that cells undergoing overt re-replication experience extensive inviability (Jallepalli et al, 1997;Yanow et al, 2001;Wilmes et al, 2004;Green and Li, 2005) or apoptosis (Vaziri et al, 2003;Thomer et al, 2004), making cell death a more likely outcome than genomic instability or tumorigenesis.…”

“…Rereplication is a potential source of genomic instability both because it produces extra copies of chromosomal segments and because it generates DNA damage and/or replication stress Zhu et al, 2004;Archambault et al, 2005;Green and Li, 2005). Re-replication has also been potentially linked to tumorigenesis by the observation that overexpression of Cdt1, which can contribute to re-replication (reviewed in Blow and Dutta, 2005), can transform NIH3T3 cells into tumorigenic cells (Arentson et al, 2002).…”

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

“…Almost all cells induced to rereplicate die, even if the CDC6 inducing rereplication is turned off quickly after induction. Green and Li (2005) have shown that rereplication leads to double-strand DNA breaks, presumably the cause of the lethality.…”