Abstract
To maintain a stable genome, it is essential that replication origins fire only once per cell cycle. The protein Geminin is thought to prevent a second round of DNA replication by inhibiting the essential replication factor Cdt1. Geminin also affects the development of several different organs by binding and inhibiting transcription factors and chromatin-remodeling proteins. It is not known if the defects in Geminin-deficient organisms are due to overreplication or to effects on cell differentiation. We previously reported that Geminin depletion in Xenopus causes early embryonic lethality due to a Chk1-dependent G(2) cell cycle arrest just after the midblastula transition. Here we report that expressing a non-Geminin-binding Cdt1 mutant in Xenopus embryos exactly reproduces the phenotype of geminin depletion. Expressing the same mutant in replication extracts induces a partial second round of DNA replication within a single S phase. We conclude that Geminin is required to suppress a second round of DNA replication in vivo and that the phenotype of Geminin-depleted Xenopus embryos is caused by abnormal Cdt1 regulation. Expressing a nondegradable Cdt1 mutant in embryos also reproduces the Geminin-deficient phenotype. In cell extracts, the nondegradable mutant has no effect by itself but augments the amount of rereplication observed when Geminin is depleted. We conclude that Cdt1 is regulated both by Geminin binding and by degradation.
Highlights
Forms on origins of replication during late G1 phase [2]
Several groups have recently reported that reinitiation of DNA synthesis occurs in Xenopus egg replication extracts when Geminin is depleted and protein degradation is inhibited with proteasome inhibitors, whereas neither treatment by itself has any effect [7, 8, 12]
The Phenotype of Geminin-depleted Embryos Is Reproduced by Expressing a Non-Geminin-binding Cdt1 Mutant—If the phenotype of Geminin depletion is caused by increased Cdt1 activity, it should be reproduced by expressing a Cdt1 mutant that is not regulated by Geminin
Summary
Several groups have recently reported that reinitiation of DNA synthesis occurs in Xenopus egg replication extracts when Geminin is depleted and protein degradation is inhibited with proteasome inhibitors, whereas neither treatment by itself has any effect [7, 8, 12]. It was not determined whether stabilization of Cdt itself was responsible for rereplication in these experiments. We sought to determine whether Geminin is required to prevent rereplication in Xenopus embryos and whether the phenotype of Geminin depletion is due to abnormal DNA replication or to an effect on cell differentiation
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