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）修复双链断裂（DSB）平衡的扰动有关。dsb在衰老和衰老细胞中积累，HR基因的纯合突变，如导致布鲁姆综合征、维尔纳综合征或范可尼贫血的基因，与早衰、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畸变和DNA断裂的积累是由DSB修复的原发性缺陷引起的，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，衰老会破坏HR控制的稳定性，以牺牲无错误的“短路基因转换”为代价，而有利于容易出错的“长路基因转换”，但在这方面，衰老的哺乳动物细胞尚未进行研究。为了解决这个问题，我们开发了一种携带新的HR报告基因的小鼠品系，可以对活细胞中的短路和长路基因转换进行快速流式细胞术分析。我们将使用这个新工具来确定小鼠的衰老是否与HR偏向于容易出错的途径（如长束基因转换）有关。我们的具体目标是：测定衰老对新鲜外植小鼠原代成纤维细胞HR质量的影响。目标2。测定衰老对新外植红系祖细胞HR质量的影响。本提案中所描述的工作的成功将揭示与衰老相关的表型的新机制。

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.