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

摘要细胞积累DNA损伤，必须修复才能保持基因组稳定。核苷酸切除修复(NER)可以消除紫外线引起的DNA损伤，当DNA损伤出现在基因转录的链上时，会阻止RNA聚合酶。NER在生物体中是保守的；我们使用酵母作为遗传上容易处理的模型来研究NER如何在体内运作。*在细胞中，DNA被埋藏在染色质中。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NER的染色质。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，降低了染色质修饰酶的参与。*自2006年以来，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。*我们提出了三个项目：*1)研究阻止的RNA聚合酶-I(RNAPI)是如何从受损的转录链中释放出来的。我们发现，延长的RNAPI在紫外线损伤时阻止，然后从转录的链中释放。我们将研究在RNAPI转录延伸和转录终止方面存在缺陷的酵母突变株中，RNAPI是如何从受损的转录链中释放出来的。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研究。*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基因座由大约150个rRNA基因组成：一半是转录的，没有核小体，另一半是转录的，有核小体的。我们将对野生型和酵母染色质修饰酶的两种结构中的NER进行比较。这些结果将有助于确定NER如何去除染色质中的DNA损伤。为了分别测定无核小体rRNA基因(内对照)和有核小体rRNA基因中的NER，我们将采用核酶切法和补骨脂素交联法对染色质进行检测。紫外线诱导DNA损伤的定位将通过T4EndoV和引物延伸分析来完成。*3)寻找染色质中与NER相关的辅助因子。然而，未知的蛋白质因素可以促进染色质中的NER。我们将采用蛋白质组学的方法，即目标基因组区域将被标记为用于定点重组的序列。在NER期间，重组将被诱导释放染色质环，这些染色质环将与德国雷根斯堡大学的Griesenbeck博士合作，通过质谱学进行分离和分析。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的理解，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修复和细胞生存的影响是什么。

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.