Mobility Control of a Yeast Retrotransposon

酵母反转录转座子的运动控制

• 批准号: 6954302
• 负责人: JILL B KEENEY
• 金额: $ 19.44万
• 依托单位: JUNIATA COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2008-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954302
• 关键词: DNA repair; Saccharomyces cerevisiae; cell motility; fungal genetics; gene deletion mutation; gene targeting; genetic recombination; oxidoreductase inhibitor; phosphorylation; ribonucleotide reductase; southern blotting; temperature sensitive mutant; transposon /insertion element

DESCRIPTION (provided by applicant): The Ty1 retroelement of yeast transposes via a mechanism bearing many similarities to retroviruses which infect larger eukaryotes. The Ty1 transposition process is inherently temperature sensitive. In a screen of the Saccharomyces deletion collection we identified RFX1 and SML1 as inhibitors of high temperature Ty1 transposition. RFX1 and SML1 are both negative regulators of ribonucleotide reductase (RNR), the rate limiting enzyme in the production of dNTPs. An adequate supply of dNTPs is essential for cellular division and DNA repair, and RNR is regulated by both of these processes. DNA repair is tightly regulated, critical for maintaining genome stability, and defects in DNA repair, as well as elevated RNR activity, is seen in some types of human cancers. The transposition process generates double stranded DNA molecules that are then integrated or recombined into the genome, a process that has been shown to involve components of the DNA repair pathway. We have identified two additional genes that regulate Ty1 mobility by inhibiting recombination of Ty1 cDNA. RFX1 and SML1 are effectors of the central Mec1 DNA repair pathway. We propose to use genetic analysis to determine the interactions of RFX1 and SML1 with other members of the Mec1 pathway in regulating Ty1 mobility. The Rad53 transducer, which acts downstream of Mec1, is an important mediator of this pathway. We will use phosphorylation of the Rad53 protein as a marker for induction of the Mec1 repair pathway during transposition. We will also attempt to determine whether Ty1 DNA sequences are recognized differently than other sequences by the cellular homologous recombination pathway. Finally, Southern analysis will be used to determine the stability and recombination targets of Ty1 cDNA molecules in various mutant strains, and the role of Ty1 integrase in these processes.

描述（由申请人提供）：酵母的Ty1逆转录元件通过与感染较大真核生物的逆转录病毒具有许多相似性的机制转座。Ty1转座过程固有地对温度敏感。在筛选的酵母缺失收集，我们确定RFX1和SML1作为高温Ty1转座的抑制剂。 RFX1和SML1都是核糖核苷酸还原酶（RNR）的负调节因子，RNR是dNTPs生产中的限速酶。充足的dNTPs供应对于细胞分裂和DNA修复至关重要，而RNR受这两个过程的调节。DNA修复受到严格调控，对于维持基因组稳定性至关重要，DNA修复缺陷以及RNR活性升高在某些类型的人类癌症中可见。转座过程产生双链DNA分子，然后将其整合或重组到基因组中，这一过程已被证明涉及DNA修复途径的组分。我们已经确定了两个额外的基因，调节Ty1流动性抑制重组Ty1 cDNA。 RFX1和SML1是中心Mec1 DNA修复途径的效应子。我们建议使用遗传分析来确定RFX1和SML1与Mec1途径的其他成员在调节Ty1流动性的相互作用。作用于Mec1下游的Rad53传感器是该通路的重要介质。我们将使用Rad53蛋白的磷酸化作为转座过程中诱导Mec1修复途径的标志物。我们还将尝试确定Ty1 DNA序列是否通过细胞同源重组途径与其他序列不同地被识别。最后，Southern分析将用于确定Ty1 cDNA分子在各种突变株中的稳定性和重组靶点，以及Ty1整合酶在这些过程中的作用。