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

总结 细胞积累DNA损伤，必须修复以维持基因组稳定性。 核苷酸切除修复（NER）去除紫外线诱导的DNA损伤，当存在于基因转录链上时，阻断RNA聚合酶。 NER在生物体中是保守的;我们使用酵母作为遗传学上易于处理的模型来研究NER在体内是如何运作的。 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，核小体的内在流动性允许DNA损伤暴露于NER，降低了染色质修饰酶的参与。自2006年以来，NSERC支持我们的研究计划是帮助描述NER如何检测和消除UV诱导的染色质DNA损伤。我们提出了3个项目：1）研究阻断的RNA聚合酶-I（RNAPI）如何从受损的转录链中释放出来。 我们发现，延长RNAPI阻断紫外线损伤，然后从转录链释放。 我们将研究RNAPI是如何从酵母突变体中受损的转录链中释放出来的，这些突变体在RNAPI转录延长和转录终止方面有缺陷。 通过染色质免疫沉淀法、染色质内源性裂解法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基因座由约150个rRNA基因形成：一半被转录，没有核小体，一半被抑制，有核小体。 我们将比较NER的两种结构，在野生型和酵母突变体的染色质修饰酶。 结果将有助于确定NER如何去除染色质中的DNA损伤。 为了分别测定无核小体的rRNA基因（内部对照）和有核小体的rRNA基因的NER，我们将在细胞核中采用限制性内切酶消化和染色质的peptien交联。 将通过T4 endoV和引物延伸测定来进行UV诱导的DNA损伤的作图。 3)在染色质中寻找与NER相关的重要因素。 然而，未知的蛋白质因子可以促进染色质中的NER。 我们将采取蛋白质组学方法，即靶基因组区域将被标记序列的位点特异性重组。 在NER过程中，重组将被诱导释放染色质环，这些染色质环将被分离并通过质谱分析进行分析，与里根斯堡大学（德国）的Griesenbeck博士合作。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如何维持，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.