MECHANISM AND REGULATION OF DNA RECOMBINATION IN SACCHAROMYCES CEREVISIAE

细胞的大部分DNA被包装成位于细胞核内的染色体。一个额外的小圆圈 只编码几个基因的基因组位于线粒体中。在某些情况下，细胞会释放 来自线粒体DNA(MtDNA)的染色体外DNA片段，来自核基因组或以 反转录转座子c DNA(RT-c DNA)。DNA片段的释放有两个后果。首先，许多生物 激活一种可能导致慢性炎症和细胞死亡的先天免疫反应。这种炎症 可能会导致严重的人类疾病，包括心力衰竭、帕金森氏症或衰老- 相关的慢性炎症。第二，释放的DNA可以插入DNA双链断裂(DSB) 导致基因组不稳定。线粒体DNA、RT-cDNA和染色体片段的插入破坏了基因组的稳定 在癌症中很常见，而V(D)J基因座上罕见的基因插入构成了抗体的途径 多元化。尽管有大量证据表明DNA碎片的释放会带来严重后果， 目前还不清楚是什么细胞事件导致了它的产生。这项建议的目标是了解 DSB中游离DNA的形成和插入机制。我们将：(I)开发一种高通量方法来 捕获和测序游离DNA，(Ii)了解DSB处插入游离DNA的机制 非同源末端连接和交替末端连接，以及(Iii)破译线性DNA的机制 从线粒体和核基因组中释放片段。

Most of a cell’s DNA is packaged into chromosomes located within the nucleus. An additional small circular genome encoding only a few genes is located in the mitochondria. In some circumstances, cells release extrachromosomal pieces of DNA from mitochondrial DNA (mtDNA), from the nuclear genome or in the form of retrotransposon cDNA (rt-cDNA). There are two consequences of DNA fragment release. First, many organisms activate an innate immune response that may result in chronic inflammation and cell death. This inflammation may lead to severe diseases in human including some cases of heart failure, Parkinson’s disease or aging- related chronic inflammation. Second, released DNA can be inserted at DNA double strand breaks (DSBs) causing genome instability. Insertions of mtDNA, rt-cDNA and chromosome fragments destabilize the genome and are common in cancer, whereas rare gene insertions at the V(D)J locus constitute a pathway of antibody diversification. Despite a large body of evidence showing severe consequences of the release of DNA fragments, it remains unknown what cellular events lead to its generation. The goal of this proposal is to understand the mechanisms of free DNA formation and its insertion at DSBs. We will: (i) develop a high throughput method to capture and sequence free DNA, (ii) understand the mechanism of insertion of free DNA at DSBs by nonhomologous end joining and by alternative end joining, and (iii) decipher the mechanisms of linear DNA fragment release from mitochondria and from the nuclear genome.