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被阻止，如果问题得不到解决，将导致细胞死亡。关于最近的学说，存在三个独立的过程来解决复制阻止：1。翻译合成的特殊dna聚合酶（TLS）， 2。与姐妹染色单体同源重组的无差错病变旁路。无错误复制后修复（PRR），其机制尚不清楚。这些机制的使用是通过修饰加工因子PCNA来调节的：赖氨酸164的单泛素化导致翻译合成，赖氨酸164的多泛素化导致无差错PRR。赖氨酸164的SUMOylation反而抑制了HR的使用。最近，数据显示人力资源部门仍在参与无差错的PRR。为此，可以提出一种包含HR的无差错PRR模型。该模型基于双重机制：一方面，在翻译合成不可能或效率低下的情况下，HR旁路病变可作为替代方案。另一方面，通过引入支持Pol eta的第二聚合酶（Pol zeta），可以提高Pol eta在TLS中的低效率。基于该模型，可以做出两种预测：1。多泛素化PCNA和2可提高HR的效率。聚泛素化PCNA可以提高Pol - zeta的效率，或者Pol - zeta被特异性地招募到聚泛素化PCNA上。这里提出的项目目的是通过使用生化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.