Abstract
Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.
Highlights
Plex (ORC)1 and/or other proteins and DNA
A previous application of a chemical genetics approach to understanding the cytotoxic effects of adozelesin suggests that the increased sensitivity of proliferating budding yeast cells to this drug depends in part on the origin recognition complex (ORC)-dependent “origin licensing” mechanism that regulates the initiation of DNA replication in proliferating cells, which is absent from cells in G0 [5]
The reduced viability of orc2-1 compared with wild type cells treated with adozelesin or methyl methanesulfonate (MMS) reported here (Fig. 1) and previously [5, 6] and the suppression of orc2-1-dependent cytotoxic effects by high levels of wild type Orc2p (Fig. 2A) clearly establish that the lethality of DNA-damaging agents is exacerbated by reduced ORC function
Summary
Plex (ORC)1 and/or other proteins and DNA (reviewed in Ref. 2). the specificity of these protein-DNA interactions varies in different organisms or at different developmental stages, they are critically important to origin function. Ectopic expression of Mge1p suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin.
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