RUI: How do the snoA and snoB Suppressors Modify Function of the Aspergillus NimODbf4 Initiator of DNA Synthesis?

在真核生物中，dbf4依赖性激酶（DDK）通过磷酸化复制DNA解旋酶Mcm2p的至少一个组分，在复制起点触发DNA合成。这反过来导致DNA的解绕和复制叉的建立。在丝状真菌中，DDK由一个调节亚基nimO Dbf4和一个激酶催化亚基cdc7asp组成。nimOp-cdc7p激酶的底物由imq Mcm2基因编码。两个新发现的基因snoA和snoB （nimo抑制因子）的突变部分减轻了nimO18突变的热敏性。snoA抑制因子通过稳定nimO18p或提高nimO18p水平间接起作用，而snoB抑制可能通过与nimO18p直接相关而发生。本项目致力于分离snoA和snoBgenes，并确定snoAp和snoBp影响nimOpfunction的分子机制。具体研究目的如下：(1)采用行走、测序和功能互补相结合的方法分离snoa和snoB基因并对其进行分子表征；(2)利用细胞生物学和生化工具研究snoA和snoB抑制因子改变nimO功能的机制。例如，体内DNA标记将用于测量DNA合成，而northern blotting将用于评估nimOmRNA水平。表位标记的nimO和/或cdc7asp等位基因将用于表征nimOpcell周期动力学，包括周转，磷酸化，定位以及snoa和snoB抑制因子的影响。nimOp-Cdc7p激酶检测将使用标记的等位基因和mcm2p nimQ作为底物。该项目将通过定义与DNA合成启动物相互作用的两个基因的功能，加强对真核生物DNA合成和细胞周期调控的理解。这些发现可能反过来为细胞分裂提供新的见解。此外，该项目将为本科生提供机会，在一个正在进行的、科学严谨的研究项目中发挥积极、实质性的作用。

In eukaryotes, the Dbf4-dependent kinase (DDK) triggers DNA synthesis at origins ofreplication by phosphorylating at least one component of the replicative DNA helicase, Mcm2p.This in turn leads to unwinding of DNA and establishment of the replication fork. In thefilamentous fungus Aspergillus nidulans, DDK is composed of a regulatory subunit, nimO Dbf4 ,and a kinase catalytic subunit, cdc7 asp . The substrate of nimOp-cdc7p kinase is encoded by thenimQ Mcm2 gene. Mutations in two newly identified genes, snoA and snoB (suppressor-of-nimO),partially alleviate the heat sensitivity of the nimO18 mutation. snoA suppressors act indirectly bystabilizing nimO18p or by elevating nimO18p levels, whereas snoB suppression may occurthrough direct association with nimO18p. This project is devoted to isolating the snoA and snoBgenes, and to defining the molecular mechanisms by which snoAp and snoBp influence nimOpfunction. The specific aims of the research are as follows: (1) use a combination of walking,sequencing, and functional complementation to isolate and molecularly characterize the snoAand snoB genes; and (2) use cell biological and biochemical tools to investigate mechanisms bywhich snoA and snoB suppressors modify nimO function. For example, in vivo DNA labelingwill be used to measure DNA synthesis, and northern blotting will be used to assess nimOmRNA levels. Epitope-tagged nimO and/or cdc7 asp alleles will be used to characterize nimOpcell cycle dynamics, including turnover, phosphorylation, localization, and the influence of snoAand snoB suppressors. A nimOp-Cdc7p kinase assay will employ the tagged alleles andMcm2p nimQ as a substrate. This project will enhance the understanding of eukaryotic DNA synthesis and cell cycle regulation by defining the functions of two genes that interact with an initiator of DNA synthesis. These discoveries may in turn provide new insights about cell division. In addition, this project will provide undergraduate students with the opportunity to play an active, substantive role in an ongoing, scientifically rigorous research project.