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Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication
M Weinberger1, P A Trabold, M Lu
1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Abstract:
DNA damaging agents induce a conserved intra-S-phase checkpoint that inhibits DNA replication in eukaryotic cells. To better understand this checkpoint and its role in determining the efficacy of antitumor drugs that damage DNA, we examined the effects of adozelesin, a DNA-alkylating antitumor agent that has a profound inhibitory effect on initiation of DNA replication in mammals, on the replication of Saccharomyces cerevisiae chromosomes. Adozelesin inhibited initiation of S. cerevisiae DNA replication by inducing an intra-S-phase DNA damage checkpoint. This inhibitory effect was abrogated in orc2-1 cells containing a temperature-sensitive mutation in a component of the origin recognition complex (ORC) that also causes a defect in initiation. The orc2-1 mutation also caused a defect in a checkpoint that regulates the activation of origins in late S phase in cells treated with hydroxyurea. Defects in both initiation and checkpoint regulation in the orc2-1 strain were suppressed by deletion of a gene encoding a putative acetyltransferase, SAS2. Adozelesin also induced a cellular response that requires a function of ORC in G(1). A similar G(1)-specific response in mammals may contribute to the cytotoxic and antitumor properties of this and other DNA-damaging drugs.
Insights
DNA damaging agents trigger a cell cycle checkpoint that halts DNA replication. This study reveals how adozelesin affects this process in yeast, identifying key genetic factors involved in DNA replication and damage response.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Damage Response
Background:
- DNA damaging agents activate conserved intra-S-phase checkpoints in eukaryotic cells, inhibiting DNA replication.
- Understanding these checkpoints is crucial for evaluating the efficacy of DNA-damaging antitumor drugs.
Purpose of the Study:
- To investigate the effects of the DNA-alkylating agent adozelesin on Saccharomyces cerevisiae chromosome replication.
- To elucidate the role of the origin recognition complex (ORC) in the intra-S-phase DNA damage checkpoint.
Main Methods:
- Treatment of Saccharomyces cerevisiae with adozelesin and hydroxyurea.
- Analysis of DNA replication initiation in wild-type and orc2-1 mutant cells.
- Genetic manipulation including gene deletion (SAS2) and temperature-sensitive mutations.
Main Results:
- Adozelesin inhibited DNA replication initiation in S. cerevisiae by inducing an intra-S-phase DNA damage checkpoint.
- The inhibitory effect of adozelesin was abolished in orc2-1 cells, which have defects in initiation and checkpoint regulation.
- Deletion of SAS2 suppressed initiation and checkpoint defects in the orc2-1 strain.
- Adozelesin induced a G(1)-specific response dependent on ORC function.
Conclusions:
- The origin recognition complex (ORC) plays a critical role in the intra-S-phase DNA damage checkpoint response to adozelesin.
- SAS2 gene deletion can rescue defects in DNA replication initiation and checkpoint control.
- A conserved G(1)-specific response involving ORC may contribute to the antitumor activity of DNA-damaging drugs in mammals.