A mouse model for studying homologous recombination fidelity during aging

描述(由申请人提供)：本提案的长期目标是确定衰老是否与同源重组(HR)引起的双链断裂(DSB)修复平衡紊乱有关。DSB在衰老和衰老的细胞中积累，HR基因的纯合子突变，如导致Bloom综合征、Werner综合征或Fanconi贫血的基因，与过早衰老、年龄相关疾病和癌症易感性有关。然而，目前尚不清楚正常衰老细胞中遗传异常和DNA断裂的积累是由DSB修复的初级缺陷引起的，还是反映了与衰老相关的继发性变化。在模式生物中的少量研究表明，衰老破坏了HR控制的稳定性，代价是无差错的“短程基因转换”，有利于容易出错的“长程基因转换”，但衰老的哺乳动物细胞尚未在这方面进行研究。为了解决这个问题，我们开发了一种携带新的HR报告的小鼠品系，它可以对活细胞中的短链和长链基因转换进行快速的流式细胞术分析。我们将使用这一新工具来确定小鼠的衰老是否与HR的偏斜有关，这些偏斜有利于长链基因转换等容易出错的途径。我们的具体目标是：目的1.确定老化对新鲜移植的原代小鼠成纤维细胞HR质量的影响。目的2.确定老化对新鲜移植的红系祖细胞HR质量的影响。这项提案中描述的工作的成功将揭示与衰老相关的表型潜在的新机制。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to determine whether aging is associated with perturbations in the balance of double strand break (DSB) repair by homologous recombination (HR). DSBs accumulate in aging and senescent cells, and homozygous mutations in HR genes such as those causing Bloom's syndrome, Werner syndrome or Fanconi anemia are associated with premature aging, age-related disease and cancer predisposition. However, it is not known whether the accumulation of genetic aberrations and DNA breaks in normal aging cells is caused by a primary defect in DSB repair or whether it reflects secondary changes associated with aging. A small number of studies in model organisms suggest that aging destabilizes HR control at the expense of error-free "short tract gene conversion" and in favor of error-prone "long tract gene conversion", but aging mammalian cells have not yet been studied in this regard. To address this question, we have developed a mouse strain carrying a new HR reporter that allows rapid flow cytometric analysis of short- and long-tract gene conversion in living cells. We will use this new tool to determine whether aging in mice is associated with skewing of HR in favor of error-prone pathways such as long tract gene conversion. Our Specific Aims are: Aim 1. Determine the impact of aging on the quality of HR in freshly explanted primary mouse fibroblasts. Aim 2. Determine the impact of aging on the quality of HR in freshly explanted erythroid progenitors. Success in the work described in this proposal will reveal new mechanisms underlying phenotypes associated with aging.