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

模式生物酿酒酵母中紫外线诱导的 DNA 损伤和染色质结构的核苷酸切除修复

• 批准号: RGPIN-2017-05495
• 负责人: Conconi, Antonio
• 金额: $ 2.04万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760924
• 关键词: Nucleotide; excision; repair; UV; induced

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.

摘要 细胞会积累 DNA 损伤，必须修复这些损伤才能维持基因组稳定性。 核苷酸切除修复 (NER) 可消除紫外线诱导的 DNA 损伤，当该损伤出现在基因转录链上时，会阻断 RNA 聚合酶。 NER 在生物体中是保守的；我们使用酵母作为遗传易处理的模型来研究 NER 在体内如何运作。在细胞中，DNA 埋藏在染色质中。 因此，NER 必须发现并消除染色质中的 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，从而降低染色质修饰酶的参与。自 2006 年以来，我们的研究计划得到 NSERC 的支持，旨在帮助描述 NER 如何检测和消除染色质中紫外线诱导的 DNA 损伤。我们提出了 3 个项目：1) 研究如何阻断 RNA 聚合酶-I (RNAAPI) 从受损的转录链中释放出来。 我们发现，延长的 RNAPI 会阻断紫外线损伤，然后从转录链中释放出来。 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体中 RNAPI 如何从受损的转录链中释放出来。 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，即由约 150 个 rRNA 基因形成的酵母 rDNA 基因座：一半被转录且没有核小体，一半被抑制且带有核小体。 我们将比较野生型和酵母突变体两种结构中染色质修饰酶的 NER。 这些结果将有助于确定 NER 如何消除染色质中的 DNA 损伤。 为了分别测量无核小体的 rRNA 基因（内部对照）和有核小体的 rRNA 基因的 NER，我们将采用细胞核限制性酶消化和染色质的补骨脂素交联。 紫外线诱导的 DNA 损伤图谱将通过 T4endoV 和引物延伸测定完成。 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能会促进染色质中的 NER。 我们将采用蛋白质组学方法，用序列标记目标基因组区域以进行位点特异性重组。 在 NER 期间，将诱导重组释放染色质环，并与雷根斯堡大学（德国）的 Griesenbeck 博士合作，通过质谱法分离和分析这些环。这些研究将培养遗传学、生物化学和蛋白质组学的研究生。 它们将增进我们对染色质 NER 的认识，有助于了解如何维持基因组稳定性，以及促进染色质 NER 的失败过程对 DNA 修复和细胞存活有何影响。