THE ROLE OF P21 IN THE REPLICATION OF DAMAGED DNA

P21 在受损 DNA 复制中的作用

基本信息

  • 批准号:
    8264928
  • 负责人:
  • 金额:
    $ 5.27万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-06-01 至 2013-06-01
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Given the mutagenic potential of DNA lesions, the mechanisms in charge of the removal of damaged bases or the elimination of genetically unstable cells constitute our main lines of defense against cancer. However, the excessive removal of cells with acceptable levels of DNA damage is also detrimental for multicellular organisms. In that context it is important to consider that the cells are particularly sensitive when DNA replication takes place since cell death can be triggered by the irreversibly stall of DNA polymerases (Pols) at DNA lesions. In such scenario auxiliary events know as DNA-damage tolerance mechanism promote DNA replication and insofar, avoid replication fork collapse and protect cell viability. A key DNA damage tolerance mechanism is called Translesion DNA synthesis (TLS). When replicative DNA Pols stop at DNA lesions, a family of specialized DNA Pols, characterized by their relaxed fidelity, can synthesize DNA opposite such lesions. In that way, the processivity of replication forks is maintained and cell death is avoided. However, TLS Pols are mutagenic when compared to replicative counterparts. Thus, it is very likely that negative regulators of TLS polymerases have the important function of inhibiting their recruitment to undamaged DNA templates in order to avoid unnecessary mutagenesis. The induction of TLS after genotoxic challenge can be studied by monitoring convenient markers such as PCNA ubiquitination, the increase in focal organization of specialized Pols and the increase in the interaction of PCNA and specialized Pols. Our data, which was obtained in collaboration with Carol Prives in Columbia University and supported by the FIRCA RO3TW007440, indicates that the cyclin kinase inhibitor p21 is a potent negative regulator of those TLS features. Interestingly, we have observed that many genotoxic treatments that require TLS activation strongly increase p21 proteolysis, being this effect dominant on p53 transactivation. Moreover, we have also observed that basal p21 levels detected in unstressed conditions also exert a negative effect on the TLS markers discussed above. Taking together our data we hypothesized that p21 might work as one important switch that represses the activity of specialized Pols on undamaged DNA but releases them to a full activation mode when DNA lesions are encountered by the replication fork. To test our hypothesis, we conceived two main aims: the first one is to determine the impact of UV-induced p21 degradation on the proper replication of damaged DNA (which depends on full TLS activation); the second aim will explore the effect of basal p21 on the control of specialized Pols activity during unstressed replication. To do so we will combing Flow Cytometry analysis with more specific techniques such as DNA combing technology, different mutagenesis assays, cytogenetic and FISH technology. Our proposal will serve to identify the exact contribution of p21 to the control of the delicate balance between DNA damage tolerance and mutagenesis. PUBLIC HEALTH RELEVANCE: DNA damage must be removed but, during the replicative phase of the cell cycle, it must also be used as a template for DNA duplication. Specialized DNA polymerases characterized by low fidelity must perform this task and therefore, they must be tightly control to avoid unnecessary mutagenesis. We have obtained preliminary evidence indicates that the p21 cyclin kinase inhibitor is a potent negative regulator of specialized DNA polymerases and it is our goal to explore its effect on the replication of damaged DNA.
描述(申请人提供):鉴于DNA损伤的突变潜力,负责移除受损碱基或消除遗传不稳定细胞的机制构成了我们对抗癌症的主要防线。然而,过度去除DNA损伤水平可接受的细胞对多细胞生物也是有害的。在这方面,重要的是要考虑到,当DNA复制发生时,细胞特别敏感,因为DNA损伤处的DNA聚合酶(POL)不可逆转地停滞可触发细胞死亡。在这种情况下,辅助事件称为DNA损伤耐受机制,促进DNA复制,并在一定程度上避免复制分叉崩溃,保护细胞存活。一种关键的DNA损伤耐受机制称为跨损伤DNA合成(TLS)。当复制型DNA POL停留在DNA损伤处时,一系列专门的DNA POL以其松弛的保真度为特征,可以合成与此类损伤相反的DNA。通过这种方式,保持了复制分叉的可处理性,并避免了细胞死亡。然而,与复制型POL相比,TLS POL具有突变性。因此,TLS聚合酶的负调控子很可能具有重要的功能,即抑制其在未受损的DNA模板上的募集,以避免不必要的突变。基因毒性攻击后TLS的诱导可以通过监测方便的标志物,如增殖细胞核抗原泛素化,专化POL的局灶化增加,以及增殖细胞核抗原和专化POL的相互作用的增加来研究。我们的数据是与哥伦比亚大学的Carol Prives合作获得的,并得到了FIRCA RO3TW007440的支持,表明周期蛋白激酶抑制剂p21是这些TLS特征的强大负调控因子。有趣的是,我们观察到许多需要TLS激活的遗传毒性治疗强烈地增加了p21蛋白的降解,这是对P53反式激活的主导作用。此外,我们还观察到,在非应激条件下检测到的基础p21水平也对上述TLS标记产生了负面影响。综合我们的数据,我们假设p21可能作为一个重要的开关发挥作用,抑制未受损DNA上专门的POL的活性,但当复制叉遇到DNA损伤时将它们释放到完全激活模式。为了验证我们的假设,我们设想了两个主要目标:第一个目标是确定紫外线诱导的p21降解对受损DNA正确复制的影响(这取决于TLS的完全激活);第二个目标是探索基础p21在非应激复制过程中控制特定POLS活性的作用。为此,我们将把流式细胞术分析与更具体的技术相结合,如DNA梳理技术、不同的诱变试验、细胞遗传学和FISH技术。我们的建议将有助于确定p21在控制DNA损伤耐受性和突变之间的微妙平衡方面的确切贡献。 与公共卫生相关:必须去除DNA损伤,但在细胞周期的复制阶段,DNA损伤也必须用作DNA复制的模板。以低保真度为特征的特殊DNA聚合酶必须执行这一任务,因此,必须严格控制它们,以避免不必要的突变。我们已经获得的初步证据表明,p21细胞周期蛋白激酶抑制物是专门化DNA聚合酶的强有力的负调控因子,我们的目标是探讨其对受损DNA复制的影响。

项目成果

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Vanesa Monica Gottifredi其他文献

Vanesa Monica Gottifredi的其他文献

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{{ truncateString('Vanesa Monica Gottifredi', 18)}}的其他基金

THE ROLE OF P21 IN THE REPLICATION OF DAMAGED DNA
P21 在受损 DNA 复制中的作用
  • 批准号:
    8077755
  • 财政年份:
    2011
  • 资助金额:
    $ 5.27万
  • 项目类别:
THE ROLE OF P21 IN THE REPLICATION OF DAMAGED DNA
P21 在受损 DNA 复制中的作用
  • 批准号:
    8485520
  • 财政年份:
    2011
  • 资助金额:
    $ 5.27万
  • 项目类别:

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