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Replication in hydroxyurea: it's a matter of time.

ABSTRACT: Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

SUBMITTER: Alvino GM

PROVIDER: S-EPMC2099622 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Replication in hydroxyurea: it's a matter of time.

Alvino Gina M GM Collingwood David D Murphy John M JM Delrow Jeffrey J Brewer Bonita J BJ Raghuraman M K MK

Molecular and cellular biology 20070716 18

Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, incl ...[more]

PMID: 17636020

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Project description:In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis in vivo and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCE Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

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Replication in hydroxyurea: it's a matter of time.

Publications

Replication in hydroxyurea: it's a matter of time.

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