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.
Highlights
Adozelesin is a member of the cyclopropylpyrroloindole (CPI)1 family of drugs, which interact with the minor groove of DNA and form covalent adducts with the N-3 of adenines
The paucity of information about this checkpoint is due to a number of factors. These include the failure to identify compounds that inhibit initiation of DNA replication, the difficulty with which trans-inhibitory effects on initiation can be distinguished from cis-inhibitory effects on the elongation of nascent DNA chains caused by lesions that block replication fork progression, and the relative difficulty of performing genetic experiments in mammalian cells compared with less complex organisms such as yeast
Adozelesin inhibited initiation of S. cerevisiae DNA replication, similar to its inhibitory effects on cellular and viral DNA replication in mammalian cells. This inhibitory effect was mediated by an intra-S-phase DNA damage checkpoint that required the function of the origin recognition complex (ORC), a highly conserved complex of proteins required for initiation of DNA replication in yeast and metazoans
Summary
Adozelesin inhibited initiation of S. cerevisiae DNA replication, similar to its inhibitory effects on cellular and viral DNA replication in mammalian cells This inhibitory effect was mediated by an intra-S-phase DNA damage checkpoint that required the function of the origin recognition complex (ORC), a highly conserved complex of proteins required for initiation of DNA replication in yeast and metazoans. Adozelesin treatment affected an ORC-dependent process in G1 that is required to maintain viability and may be related to origin licensing pathways Alterations in this process may explain the potent G1-specific cytotoxic effects of this drug in both yeast and mammals [11, 12] as well as the antitumor properties of this and related DNA damaging drugs
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