GL CYCLINS AND THE REGULATION OF DNA REPLICATION

细胞分裂和生长控制的调节对所有 有机体 细胞周期蛋白依赖性激酶（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。 许多生长刺激 和抑制信号影响CDK的活性;此外，突变 其使CDK表达失调或影响CDK功能 抑制剂与生长控制和癌症的改变有关。 已知CDK是蛋白激酶，但很少有CDK的底物具有 被识别。 这项建议利用了遗传， 分子和生物化学方法来识别和表征 Cdc 28的底物和调节剂，酿酒酵母中的CDK， 专注于DNA的调节和保真度所需的那些 合成. G1期的DNA损伤导致“检查点”反应;细胞延迟转运 直到损伤修复 CDC的作用28 将通过检查以下因素的影响来确定这种反应中的激酶 G1细胞周期蛋白（CLN）的过表达和缺失。 CLN的调节子和CLN功能下游所需的基因， 将鉴定START后DNA合成的促进作用， 特点，采用三种不同的方法。 第一种方法 从观察到检查点基因，mec 1 -1， 和GAL-CLN 1是合成致死的。 这表明， CLN 1的组成性水平可以超越DNA损伤的G1检查点， 或者GAL-CLN 1可以促进部分缺陷的DNA合成。 将选择这种合成致死性的抑制剂， 在基因、分子和生物化学上都有特征。 第二 一种方法继续分析ERC 11，其中影响DNA的突变 合成和修复。 DNA合成中的缺陷被 CLN 1或CLN 2的表达，但不表达CLN 3，表明CLN 1/CLN 2 与CLN 3不同地促进细胞周期的转运。 到 描述CLN 1/CLN 2的这种能力，我们将描述两个 erc 11、SEL 1和CDC 9的抑制子，其可以编码 CLN 1/CLN 2激活的DNA复制途径。 最后，新的cln 2突变体 在erc 11 -2突变体中不能促进DNA合成的等位基因将被 与世隔绝 这些突变的cln 2等位基因将被生物化学表征， 从基因上来确定它们是否影响了cln 2的一个子集， 功能协调发展的

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.