Regulation of Trypanosome DNA Replication

锥虫 DNA 复制的调控

基本信息

批准号：
7887941
负责人：
DAN S RAY
金额：
$ 8.04万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-03 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7887941
关键词：
Address Affinity Chromatography Binding Binding Proteins Biochemical Cell Cycle Cells Code Complex Consensus Sequence Crithidia fasciculata DNA DNA Excision Repair Protein ERCC-6 DNA Ligases DNA biosynthesis DNA-Directed DNA Polymerase Family Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Techniques Grant Half-Life Hand Immunoprecipitation Individual Kinetoplast DNA Ligase Mass Spectrum Analysis Messenger RNA Methods Mitochondrial DNA Modeling Molecular Molecular Genetics Organism Orthologous Gene Phase Plasmids Point Mutation Polymerase Protein Binding Protein Subunits Proteins Protozoa RNA Interference RNA-Binding Proteins Reaction Regulation Replication Origin Research Reverse Transcriptase Polymerase Chain Reaction Role Specificity Structure Trans-Activators Transcript Transcriptional Regulation Trypanosoma Trypanosoma brucei brucei Work cis acting element genome database in vivo knock-down novel protein complex protein metabolism repaired

项目摘要

The proposed research addresses two related questions: (1) What are the molecular mechanisms that regulate S-phase gene expression in Trypanosoma brucei? and (2) How is the expression of a specific mitochondrial DNA ligase regulated during the cell cycle and how does its expression contribute to kinetoplast structure and division? There appears to be little or no significant transcriptional regulation of protein coding genes in kinetoplastids. Rather, regulation of gene expression occurs largely at a post-transcriptional level. In earlier work with C. fasciculata we identified an octamer consensus sequence required for S-phase expression of several genes and also two protein complexes that bind to the octamer sequence with high specificity. Genes encoding these proteins are present in T. brucei as well as additional related genes that have not been investigated. We have successfully synchronized T. brucei and will use synchronized cultures to investigate S-phase gene expression and to identify and characterize the specific binding proteins. Subunits of these proteins will be identified using immunological methods and mass spectrometry. Protein complexes will also be purified and characterized using tandem affinity purification. RNA interference methods will be used to knock down expression of each of the binding proteins and to determine their importance in sequence-specific binding to the octamer sequence in transcripts. We will also use immunoprecipitation and RT-PCR to directly demonstrate the binding in vivo of these protein complexes to transcripts containing the octamer sequences. The C. fasciculata DNA ligase ka (LIG ka) is an unstable protein that shows cyclic expression and cyclic localization to the kinetoplast. We will examine the roles of octamer-related sequences in the cyclic expression of LIG ka and the adjacent LIG k¿ gene through point mutation of individual octamer-related sequences alone and in combination. These studies will address the possible effect of the cycling of each gene transcript on that of the other. We will also investigate the possible role of LIG ka in the regulated repair of gaps that remain at minicircle replication origins prior to their closure as a prelude to network division. The structure of the kDNA networks and of the network minicircles will be determined in T. brucei cells expressing LIG ka? at a constant and regulated level throughout the cell cycle. Since DNA ligases have been found to interact with DNA polymerases in gap repair reactions we will identify the specific mitochondrial DNA polymerase that might function together with LIG ka? in gap repair and will examine the ability of the ligase-polymerase complex to repair a model minicircle gap at the replication origin.

拟议的研究解决了两个相关问题：（1）调节哪些分子机制布鲁氏锥虫中的S期基因表达？（2）特定线粒体的表达如何在细胞周期中调节的DNA连接酶，其表达如何促进动力学结构和分配？在动力质体中，蛋白质编码基因的转录调节似乎很少或没有明显的转录调节。相反，基因表达的调节主要发生在转录后水平。在较早的工作中 C. fasciculata我们确定了几个基因S期表达所需的八聚体共识序列以及具有高特异性与八聚体序列结合的两个蛋白质复合物。编码这些的基因蛋白质存在于T. brucei中，以及尚未研究的其他相关基因。我们有成功同步的T. brucei，并将使用同步培养物研究S期基因表达并识别和表征特定的结合蛋白。这些蛋白质的亚基将使用免疫学方法和质谱法。蛋白质复合物也将被纯化并表征使用串联亲和力纯化。 RNA干扰方法将用于击倒每一个的表达结合蛋白并确定其在序列特异性结合中的重要性在成绩单中。我们还将使用免疫沉淀和RT-PCR直接证明体内结合这些蛋白质复合物与包含八聚体序列的转录本。 C. fasciculata DNA连接酶Ka（LIG KA）是一种不稳定的蛋白质，显示了环状表达和环状定位到动力学成形素。我们将检查八聚体相关序列在环状表达中的作用通过单独的八聚体相关序列的点突变，LIG KA和相邻的LIGk¿基因并结合在一起。这些研究将解决每个基因转录本循环的可能影响另一个。我们还将调查LIG KA在保留位置的间隙修复中的可能作用微小复制起源是在其关闭之前作为网络部门的序幕。 kDNA的结构网络和网络小圆圈将在表达LIG KA的T. brucei细胞中确定？保持不变并在整个细胞周期中受到调节水平。由于已经发现DNA连接酶与DNA相互作用间隙修复反应中的聚合酶我们将确定可能的线粒体DNA聚合酶与lig ka一起功能？在间隙修复中，将检查连接酶 - 聚合酶复合物的能力修复模型的小节间隙，处于复制起源。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mitochondrial DNA ligase in Crithidia fasciculata.

DOI：
10.1073/pnas.0305705101
发表时间：
2004-03
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
K. Sinha;J. C. Hines;N. Downey;D. Ray
通讯作者：
K. Sinha;J. C. Hines;N. Downey;D. Ray

RNA primer removal and gap filling on a model minicircle replication intermediate.

模型小环复制中间体上的 RNA 引物去除和间隙填充。