AGING EFFECTS ON DNA REPAIR

衰老对 DNA 修复的影响

基本信息

批准号：
2667623
负责人：
Glenn Wilson
金额：
$ 20.78万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-04-01 至 2000-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2667623
关键词：
DNA repair DNA replication aging antibody bromodeoxyuridine density gradient ultracentrifugation methylpurine methyltransferase mitochondrial DNA nitrogen mustard oxidative stress polymerase chain reaction purines tissue /cell culture

项目摘要

This proposal has been structured to help test the hypothesis that it is the accumulation of damage within DNA that causes some of the phenotypes associated with aging. Specifically, the objective of this application is to determine how DNA repair mechanisms change with aging. Repair of DNA in both the nucleus and the mitochondria will be evaluated in various human cell cultures which are models of aging. The objective of the proposal will be pursued through the following specific aims. i) An examination of the effects of aging on the repair of oxidative damage in DNA. These experiments will evaluate the repair of sugar-phosphate and base lesions caused by reactive oxygen species in both mitochondrial DNA and specific nuclear sequences. 2) A study of the effects of aging on the repair on N- methylpurines. These studies will evaluate the repair of N7-methylguanine and N3-methyladenine in both mitochondrial DNA and specific nuclear sequences. 3) An evaluation of the effects of aging on the repair of 06- methylguanine. Initial studies in this aim will correlate the repair of this mutagenic lesion in the total genome with the levels of 06- methylguanine-DNA-methyltransferase in the cells. Subsequent studies will employ an antibody specific to this lesion and PCR technology to determine the repair of this DNA adduct in mitochondrial DNA and specific transcribed and nontranscribed sequences of nuclear DNA. 4) A determination of the effects of aging on the replication of mitochondrial DNA. These studies will use the incorporation of the heavy base analogue bromodeoxyuridine into DNA during replication. This makes it possible to separate replicated mitochondrial DNA from nonreplicated DNA on cesium chloride gradients so that DNA replication can be quantified. Both the effects of aging alone and in combination with exposure to nitrogen mustard, which produces adducts which are not repaired in mitochondrial DNA, on mitochondrial DNA replication will be studied. When successfully completed,. these studies will provide a more thorough understanding of how the aging process can adversely affect DNA repair mechanisms and cause the accumulation of DNA damage which would lead to impairment of normal cellular functions and/or death.

该提案的结构是为了帮助检验以下假设： DNA 内损伤的累积导致某些表型与衰老有关。具体来说，该应用程序的目标是确定 DNA 修复机制如何随衰老而变化。 DNA 的修复细胞核和线粒体都将在不同的人体中进行评估细胞培养物是衰老模型。提案的目的将通过以下具体目标来实现。 i) 检查衰老对 DNA 氧化损伤修复的影响。这些实验将评估糖磷酸盐和碱基损伤的修复由线粒体DNA和特定的活性氧引起核序列。 2) 老化对N-修复影响的研究甲基嘌呤。这些研究将评估 N7-甲基鸟嘌呤的修复线粒体 DNA 和特定核中的 N3-甲基腺嘌呤序列。 3) 老化对06-修复影响的评估甲基鸟嘌呤。这一目标的初步研究将与修复相关总基因组中的这种诱变损伤的水平为 06- 细胞中的甲基鸟嘌呤-DNA-甲基转移酶。后续研究将使用针对该病变的特异性抗体和 PCR 技术来确定线粒体 DNA 中 DNA 加合物的修复和特异性核DNA的转录和非转录序列。 4) 一个确定衰老对线粒体复制的影响脱氧核糖核酸。这些研究将使用重碱类似物的掺入复制过程中溴脱氧尿苷进入 DNA。这使得可以在铯上将复制的线粒体 DNA 与非复制的 DNA 分离氯化物梯度，以便可以量化 DNA 复制。两者都单独老化以及与暴露于氮气结合的老化的影响芥末，产生的加合物在线粒体中无法修复 DNA，对线粒体DNA复制将进行研究。成功时完全的，。这些研究将提供更全面的了解衰老过程如何对 DNA 修复机制产生不利影响并导致 DNA损伤的积累会导致正常功能受损细胞功能和/或死亡。