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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis
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Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis

机译：酵母增殖细胞核抗原的泛素化及其对转移DNA合成的影响

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The Rad6-Rac118 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) eta- and zeta-depenclent translesion DNA synthesis (TLS) and a Rad5-Mms2-Ubc13-dependent pathway. In DNA-damaged yeast cells proliferating cell nuclear antigen (PCNA) becomes monoubiquitylated at the K164 residue, and genetic studies in yeast have indicated a requirement for this modification in TILS mediated by Pol eta and Pol zeta. To be able to decipher the role of PCNA monoubiquitylation in the TILS process, we have reconstituted this PCNA modification in vitro from purified yeast proteins. We show that, in addition to the requirement for Rad6-Rad18, the reaction depends on the loading of the PCNA homotrimeric ring onto the DNA by replication factor C and that all three PCNA monomers become efficiently ubiquitylated. The availability of PCNA monoubiquitylated on all of its three monomers has enabled us to examine the effects of this PCNA modification on DNA synthesis by Pols delta, eta, zeta, and Rev1. Contrary to the prevailing ideas that presume a role for PCNA ubiquitylation in the disruption of Pol delta's binding to PCNA or in the enhancement of the binding affinity of the TLS Pots for PCNA, we find that PCNA ubiquitylation does not affect any of these processes. These observations lead us to suggest a role for PCNA monoubiquitylation in disrupting the PCNA binding of a protein(s) that otherwise is inhibitory to the binding of PCNA by TLS Pols.
机译：Rad6-Rac118泛素结合酶复合物通过至少三种不同途径促进通过DNA损伤的复制：DNA聚合酶（Pol）eta和zeta依赖的转移DNA合成（TLS）和Rad5-Mms2-Ubc13依赖性途径。在DNA损伤的酵母细胞中，增殖细胞核抗原（PCNA）在K164残基处被单泛素化，并且酵母中的遗传研究表明需要对Pol eta和Pol zeta介导的TILS进行这种修饰。为了能够解释PCNA单泛素化在TILS过程中的作用，我们从纯化的酵母蛋白中体外重建了PCNA修饰。我们表明，除了对Rad6-Rad18的要求外，反应还取决于复制因子C将PCNA同源三聚体环装载到DNA上，并且所有三个PCNA单体都被有效地泛素化了。在其所有三种单体上单泛素化的PCNA的可用性使我们能够研究Pols delta，eta，zeta和Rev1的PCNA修饰对DNA合成的影响。与认为PCNA泛素化在破坏Pol delta与PCNA的结合或增强TLS Pots对PCNA的结合亲和力中起作用的流行观点相反，我们发现PCNA泛素化不影响任何这些过程。这些观察结果使我们提出PCNA单泛素化在破坏蛋白质的PCNA结合中的作用，否则该蛋白质会抑制TLS Pols对PCNA的结合。

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