THE ROLE OF P21 IN THE REPLICATION OF DAMAGED DNA

描述（由申请人提供）：鉴于DNA损伤的致突变潜力，负责去除受损碱基或消除遗传不稳定细胞的机制构成了我们抵御癌症的主要防线。然而，过度去除具有可接受水平DNA损伤的细胞对多细胞生物也是有害的。在这种情况下，重要的是要考虑到，当DNA复制发生时，细胞是特别敏感的，因为DNA损伤处DNA聚合酶（pol）的不可逆停滞可能引发细胞死亡。在这种情况下，被称为DNA损伤耐受机制的辅助事件促进DNA复制，从而避免复制分叉崩溃，保护细胞活力。一个关键的DNA损伤耐受机制被称为翻译DNA合成（TLS）。当复制的DNA pol停在DNA损伤处时，一个具有松弛保真度的特化DNA pol家族可以在损伤处合成DNA。通过这种方式，保持了复制分叉的进程性，避免了细胞死亡。然而，与复制对等体相比，TLS poll具有诱变性。因此，TLS聚合酶的负调节因子很可能具有抑制其向未受损DNA模板募集以避免不必要的突变的重要功能。通过监测PCNA泛素化、特异性pol的局灶组织增加以及PCNA与特异性pol相互作用增加等方便标记，可以研究遗传毒性攻击后TLS的诱导。我们的数据是与哥伦比亚大学的Carol Prives合作获得的，并得到FIRCA RO3TW007440的支持，表明细胞周期蛋白激酶抑制剂p21是这些TLS特征的有效负调控因子。有趣的是，我们已经观察到许多需要激活TLS的基因毒性治疗强烈地增加了p21蛋白水解，这种作用在p53反激活中占主导地位。此外，我们还观察到，在非胁迫条件下检测到的基础p21水平也会对上述TLS标记产生负面影响。综合我们的数据，我们假设p21可能作为一个重要的开关，抑制未受损DNA上特殊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适当复制的影响（这取决于TLS的完全激活）；第二个目标将探索基础p21在非应激复制过程中对特异性pol活性控制的影响。为此，我们将流式细胞术分析与更具体的技术相结合，如DNA梳理技术、不同的诱变分析、细胞遗传学和FISH技术。我们的建议将有助于确定p21对控制DNA损伤耐受性和诱变之间微妙平衡的确切贡献。

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.