Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is inappropriately re-replicated, but the importance of this stringent control on genome stability has not been tested. Here we show that re-replication in Saccharomyces cerevisiae can strongly induce the initial step of gene amplification, increasing gene copy number from one to two or more. The resulting amplicons consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. These re-replication–induced gene amplifications are mediated by nonallelic homologous recombination between the repetitive elements. We suggest that re-replication may be a contributor to gene copy number changes, which are important in fields such as cancer biology, evolution, and human genetics.
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.
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...
Objective. The authors used data from a larger study to evaluate the longterm effects of a peer advocate intervention on condom and contraceptive use among HIV-infected women and women at high risk for HIV infection.Methods. HlV-infected women in one study and women at high risk for HIV infection in a second study were selected from the Women and lnfants Demonstration Project and assigned to a standard or an enhanced HIV prevention treatment group. The enhanced intervention included support groups and one-on-one contacts with peer advocates tailored to clients' needs, The authors interviewed women at baseline and at 6-, l2-and I Bmonths, and measured changes in consistency of condom and contraceptive use and in self-efficacy and perceived advantages and disadvantages of condom and contraceptive use.Results. Of HIV-infected women, the enhanced group had improved consistency in condom use, increased perceived advantages of condom use, and increased level of self-efflcacy compared with the standard group. Of women at risk, the enhanced intervention group at six months maintained consistent condom use with a main partner and perceived more benefit of condom use compared with the standard group. These differences diminished at l2 months.Conclusions. The enhanced intervention was generally effective in the HIV+ study. In the at-risk study, however, intervention effects were minimal and shorl-lived. Factors related to the theory, intervention design, and sample characteristics help explain these differences. PUBLIC HEALTH REPORTS .2OoISUPPLEMENT I . VoLUME I16
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.