Biochemical Investigation on the Mechanism of Error Free Postreplication Repair (PRR) using baker yeast Saccharomyces cerevisiae as a model organism

DNA损伤可导致复制DNA聚合酶在复制过程中的停滞，这将导致细胞死亡，如果它仍然没有得到解决。关于最近的学说，存在三个独立的过程来解决复制逮捕：1。通过专门的DNA聚合酶（TLS）进行翻译合成，2.通过与姐妹染色单体同源重组的无错误损伤旁路和3。无差错的复制后修复（PRR），其机制仍然未知。这些机制的使用是通过修改的持续合成因子PCNA调节：单泛素化的赖氨酸164导致translesion合成，多泛素化的赖氨酸164导致错误的PRR。 赖氨酸164的SUMO化反而抑制了HR.Recently的使用，数据显示HR还参与了无错误的PRR。关于这一点，可以提出一个新的无错误PRR模型，包括HR。该模型是基于一个双重机制：一方面，病变旁路HR作为一种替代，在跨病变合成证明是不可能的或效率低下的情况下。另一方面，可以通过招募在TLS中支持Pol eta的第二聚合酶（Pol zeta）来增强Pol eta的低效TLS。基于这个模型，可以做出两个预测：1。多聚泛素化的PCNA和2. Pol zeta的效率可以通过多聚泛素化的PCNA增强，或者Pol zeta被特异性地募集到多聚泛素化的PCNA。本文提出的项目的目的是通过使用生物化学方法来测试这些预测以验证新模型。

DNA lesions can result in an arrest of the replicative DNA-polymerase during replication, which would lead to cell death in case it remains unresolved. Regarding the recent doctrine, three independent processes exist to resolve replication arrests: 1. Translesion synthesis by a specialized DNA-Polymerase (TLS), 2. Error free lesion bypass by homologous recombination with the sister chromatid and 3. Error free Postreplication Repair (PRR) whose mechanisms is still unkown. Usage of these mechanisms is regulated through modification of the processivity factor PCNA: mono-ubiquitylation of lysine 164 results in translesion synthesis, poly-ubiquitylation of lysine 164 results in error free PRR. SUMOylation of lysine 164 instead represses the usage of HR. Recently, data was shown suggesting that HR yet is participating in error free PRR. Regarding this, a new model for error free PRR may be proposed including HR. This model is based upon a dual mechanism: one the one hand, lesion bypass by HR acts as an alternative, in case translesion synthesis proves impossible or inefficient. On the other hand, inefficient TLS by Pol eta may be enhanced by recruiting a second Polymerase (Pol zeta) which supports Pol eta in TLS. Based on this model, two predictions may be made: 1. Efficiency of HR may be enhanced by poly-ubiquitylated PCNA and 2. Efficiency of Pol zeta may be enhanced by poly-ubiquitylated PCNA or Pol zeta is recruited specifically to poly-ubiquitylated PCNA. Aim of the project proposed here is to test these predictions to verify the new model by using biochemical approaches.