Gopalakrishnan V, Simancek P, Houchens C, Snaith H A, Frattini M G, Sazer S, Kelly T J
Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21210, USA.
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):13114-9. doi: 10.1073/pnas.221467598. Epub 2001 Oct 23.
The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle. We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18(CDK)) is not sufficient to induce rereplication. Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2). When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation. However, constitutive expression of Cdt1, together with Cdc18(CDK), is sufficient to induce extra rounds of DNA replication in the absence of mitosis. Significantly greater levels of rereplication can be induced by coexpression of Cdc18(CDK) and a Cdt1 mutant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other. Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability. Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.
真核细胞中复制起点处DNA复制的起始受到严格控制，以确保基因组在每个细胞周期仅复制一次。我们提供的证据表明，在裂殖酵母中，起始复合物的两个必需组分Cdc18和Cdt1的独立调节有助于防止DNA复制的重新起始。Cdc18受细胞周期蛋白依赖性激酶（CDK）磷酸化的负调控，但缺乏CDK磷酸化位点的Cdc18突变体形式（Cdc18(CDK)）的低水平表达不足以诱导重新复制。与Cdc18相似，Cdt1在细胞周期中周期性表达，在G(1)期在细胞核中积累，在G(2)期下降。当Cdt1从异位启动子组成型表达时，它在整个细胞周期中在细胞核中积累，但不促进重新起始。然而，Cdt1与Cdc18(CDK)的组成型表达足以在没有有丝分裂的情况下诱导额外的DNA复制轮次。通过共表达Cdc18(CDK)和缺乏保守C末端基序的Cdt1突变体可以诱导明显更高水平的重新复制。相反，当任何一种突变蛋白在没有另一种的情况下表达时，不会发生不受控制的DNA复制。野生型或突变型Cdt1的组成型表达也导致Cdc18(CDK)水平的增加，这可能是由于蛋白质稳定性增加的结果。我们的数据与以下假设一致，即重新复制的控制依赖于一种冗余机制，其中Cdt1的负调控与Cdc18的负调控并行发挥作用。
