Haracska Lajos, Unk Ildiko, Prakash Louise, Prakash Satya
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.
Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6477-82. doi: 10.1073/pnas.0510924103. Epub 2006 Apr 12.
The Rad6-Rad18 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) eta- and zeta-dependent 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 TLS mediated by Poleta and Polzeta. To be able to decipher the role of PCNA monoubiquitylation in the TLS 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 Poldelta's binding to PCNA or in the enhancement of the binding affinity of the TLS Pols 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.
Rad6-Rad18泛素结合酶复合物通过至少三种不同途径促进DNA损伤后的复制：DNA聚合酶（Pol）η和ζ依赖性跨损伤DNA合成（TLS）以及Rad5-Mms2-Ubc13依赖性途径。在DNA受损的酵母细胞中，增殖细胞核抗原（PCNA）在K164残基处发生单泛素化，酵母中的遗传学研究表明，这种修饰是Polη和Polζ介导的TLS所必需的。为了能够解读PCNA单泛素化在TLS过程中的作用，我们利用纯化的酵母蛋白在体外重建了这种PCNA修饰。我们发现，除了需要Rad6-Rad18外，该反应还依赖于复制因子C将PCNA同三聚体环加载到DNA上，并且所有三个PCNA单体都能有效地发生泛素化。所有三个单体都发生单泛素化的PCNA的可得性使我们能够研究这种PCNA修饰对Polδ、η、ζ和Rev1的DNA合成的影响。与普遍认为PCNA泛素化在破坏Polδ与PCNA的结合或增强TLS聚合酶与PCNA的结合亲和力中起作用的观点相反，我们发现PCNA泛素化不会影响这些过程中的任何一个。这些观察结果使我们提出，PCNA单泛素化在破坏一种蛋白质与PCNA的结合中起作用，否则这种蛋白质会抑制TLS聚合酶与PCNA的结合。
