Nucleotide excision repair of UV induced DNA damage and chromatin structure in the model organism S. cerevisiae

细胞累积的DNA损伤必须被修复以维持基因组的稳定。核苷酸切除修复（NER）去除紫外线诱导的DNA损伤，当基因转录链上存在时，阻断RNA聚合酶。NER在整个生物体中是保守的；我们使用酵母作为遗传可处理的模型来研究NER如何在体内运作。在细胞中，DNA隐藏在染色质中。因此，NER必须发现并去除染色质中的DNA损伤。对于基因组的短区域，研究表明参与DNA转录和复制的染色质修饰酶也有助于染色质中的NER。然而，有迹象表明核小体的固有移动性允许DNA损伤暴露于NER，降低染色质修饰酶的参与。******自2006年以来由NSERC支持，我们的研究计划是帮助描述NER如何检测和去除紫外线诱导的染色质DNA损伤。******我们提出三个项目：***1)研究被阻断的RNA聚合酶i （RNAPI）如何从受损的转录链中释放出来。我们发现，伸长的RNAPI在紫外线损伤下阻滞，然后从转录链上释放出来。我们将研究在RNAPI转录延伸和转录终止缺陷的酵母突变体中，RNAPI是如何从受损的转录链中释放出来的。研究将通过染色质免疫沉淀，染色质内源切割和电子显微镜进行。***2)确定参与基因转录的染色质修饰酶是否促进NER。我们有一个独特的模型，酵母rDNA位点由大约150个rRNA基因组成：一半是转录的，没有核小体，一半是抑制的，有核小体。我们将比较野生型和酵母突变体在两种结构中的染色质修饰酶。结果将有助于确定NER如何去除染色质中的DNA损伤。为了在没有核小体的rRNA基因（内控）和有核小体的rRNA基因中分别测量NER，我们将采用核内切酶酶切和染色质的补骨脂素交联。紫外诱导DNA损伤的定位将通过T4endoV和引物延伸试验来完成。***3)寻找染色质中与NER相关的辅助因子。然而，未知的蛋白质因子可能促进染色质中的NER。我们将采用蛋白质组学方法，即目标基因组区域将被标记为特定位点重组的序列。与德国雷根斯堡大学的Griesenbeck博士合作，在NER期间，将诱导重组释放染色质环，这些染色质环将被分离并通过质谱分析。******这些研究将形成遗传学，生物化学和蛋白质组学的研究生。它们将促进我们对染色质中NER的认识，将有助于理解基因组稳定性是如何维持的，以及促进染色质中NER的失败过程对DNA修复和细胞存活的影响。

SUMMARY Cells accumulate DNA damage that must be repaired to maintain genome stability. Nucleotide Excision Repair (NER) removes UV induced DNA damage that, when present on gene transcribed strands, block RNA polymerases. NER is conserved across organisms; we use yeast as genetically tractable model to study how NER operates in vivo.***In cells, DNA is buried in chromatin. Thus, NER must find and remove DNA damage in chromatin. For short regions of the genome, it was shown that chromatin modifying-enzymes that participate in DNA transcription and replication also help NER in chromatin. However, there are indications that the intrinsic mobility of nucleosomes permits exposure of DNA damage to NER, downgrading the participation of chromatin modifying-enzymes.******Supported by NSERC since 2006, our research program is to help describing how NER detects and removes UV induced DNA damage in chromatin.******We propose 3 projects:***1) To investigate how blocked RNA polymerase-I (RNAPI) is released from the damaged transcribed strand. We showed that elongating RNAPI block at UV damage and then are released from the transcribed strand. We will investigate how RNAPI are released from the damaged transcribed strand in yeast mutants that have defects in RNAPI transcription elongation and transcription termination. Investigations will be done by Chromatin Immuno-Precipitation, Chromatin Endogenous-Cleavage and Electron Microscopy.***2) To define if chromatin modifying-enzymes that participate in gene transcription promote NER. We have a unique model, the yeast rDNA locus that is formed by ~150 rRNA genes: half are transcribed and without nucleosomes and half are repressed and with nucleosomes. We will compare NER in the two structures, in wild-type and yeast mutants for chromatin modifying-enzymes. The results will help defining how NER removes DNA damage in chromatin. To measure NER separately in rRNA genes without nucleosomes (internal control) and rRNA genes with nucleosomes, we will employ in nuclei restriction enzyme-digestion and psoralen crosslinking of chromatin. Mapping of UV induced DNA damage will be done by T4endoV and primer extension assays. ***3) To search auxiliary-factors associated with NER in chromatin. Yet unknown protein factors could promote NER in chromatin. We will take a proteomic approach, whereby target genomic regions will be tagged with sequences for site-specific recombination. During NER, recombination will be induced to release chromatin rings that will be isolated and analyzed by mass spectrometry, in collaboration with Dr Griesenbeck at the University of Regensburg (Germany).******Theses studies will form graduate students in genetics, biochemistry and proteomics. They will advance our perception of NER in chromatin, will help understanding how genome stability is maintained and what is the impact of failed processes that promote NER in chromatin on DNA repair and cell survival.