GL CYCLINS AND THE REGULATION OF DNA REPLICATION

细胞分裂和生长控制的调节对ALL至关重要 有机体。细胞周期蛋白依赖性蛋白激酶(CDK)是细胞周期蛋白的重要调节因子 所有真核生物的细胞周期分裂。许多促进增长的因素 抑制信号影响CDKs的活性；此外，突变 哪些对CDK的表达有抑制作用或影响CDK的功能 抑制剂与生长控制和癌症的改变有关。 CDK是已知的蛋白激酶，但很少有CDK的底物具有 已被确认身份。这一提议利用了基因、 鉴定和表征的分子和生化方法 酿酒酵母中的CDK--CDC28的底物和调节子， 重点关注DNA的调节和保真度所需的那些 综合。 G1期的DNA损伤会引起“检查点”反应；细胞延迟转运 通过细胞周期，直到损伤修复。CDC28的作用 这种反应中的激酶将通过检查以下因素的影响来确定 G1期细胞周期蛋白(CLN)的过度表达和缺失。 CLN的调节器和CLN下游所需的基因 将确定START后促进DNA合成的情况，并 利用三种不同的方法来确定其特征。第一种方法 根据观察，检查点基因的突变Mec1-1， 和Gal-CLN1是合成致命性的。这表明，高， CLN1的构成水平可能会覆盖DNA损伤的G1检查点， 或者，Gal-CLN1可能会促进部分有缺陷的DNA合成。 这种合成杀伤力的抑制者将被选择并 在基因、分子和生物化学方面具有特征。第二个 方法继续分析ERC11，其中的突变影响DNA 合成和修复。DNA合成中的缺陷被抑制 CLN1或CLN2的表达，而不是CLN3的表达，表明CLN1/CLN2 以不同于CLN3的方式促进细胞周期的转运。至 描述CLN1/CLN2的这种能力，我们将描述两个 ER11、SEL1和CDC9的抑制子，它们可能编码 CLN1/CLN2-激活的DNA复制途径。最后，新的cln2突变体 在erc11-2突变体中不能促进DNA合成的等位基因将是 与世隔绝。这些突变的cln2等位基因将被生化鉴定。 并从基因上确定它们是否影响Cln2‘S的子集 功能。

The regulation of cell division and growth control is critical for all organisms. Cyclin-dependent kinases (CDKs) are essential regulators of cell cycle division in all eukaryotic organisms. Many growth-stimulatory and inhibitory signals affect the activity of CDKs; furthermore, mutations which deregulate expression of CDKs or affect the function of CDK inhibitors are associated with alterations in growth control and cancers. The CDKs are known to be protein kinases, but few substrates of CDKs have been identified. This proposal utilizes a combination of genetic, molecular and biochemical approaches to identify and characterize substrates and regulators of Cdc28, the CDK in Saccharomyces cerevisiae, focusing on those required for the regulation and fidelity of DNA synthesis. DNA damage in G1 causes a "checkpoint" response; the cell delays transit through the cell cycle until damage is repaired. The role of the Cdc28 kinase in this response will be determined by examining the effects of both overexpression and deletion of the G1 cyclins (CLNs). Regulators of the CLNs and genes required downstream of CLN function for promotion of DNA synthesis after START will be identified and characterized, utilizing three different approaches. The FIRST approach follows from the observation that a mutation in a checkpoint gene, mec1-1, and GAL-CLN1 are synthetically lethal. This suggests that high, constitutive levels of CLN1 may override a G1 checkpoint for DNA damage, or that GAL-CLN1 may promote partially defective DNA synthesis. Suppressors of this synthetic lethality will be selected and characterized genetically, molecularly and biochemically. The SECOND approach continues the analysis of ERC11, mutations in which affect DNA synthesis and repair. The defects in DNA synthesis are suppressed by the expression of CLN1 or CLN2, but not by CLN3, demonstrating that CLN1/CLN2 promote transit through the cell cycle differently than CLN3. To characterize this ability of CLN1/CLN2, we will characterize two suppressors of erc11, SEL1 and CDC9, which may encode components of a CLN1/CLN2-activated pathway for DNA replication. FINALLY, new cln2 mutant alleles unable to promote DNA synthesis in erc11-2 mutants will be isolated. These mutant cln2 alleles will be characterized biochemically and genetically to determine whether they affect a subset of cln2's functions.