Identification of two functional PCNA-binding domains in human DNA polymerase κ.
Identification of two functional PCNA-binding domains in human DNA polymerase κ.
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人类 DNA 聚合酶 γ 中两个功能性 PCNA 结合域的鉴定。
DOI：
10.1111/gtc.12156
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Prakash,Louise
中科院分区：
文献类型：
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作者：
Yoon,Jung-Hoon;Acharya,Narottam;Park,Jeseong;Basu,Debashree;Prakash,Satya;Prakash,Louise
以前，我们已经证明人类DNA聚合酶(Pol)η有两个功能性的增殖细胞核抗原结合基序，PIP1和PIP2，并且Polη的C端缺失，缺少泛素结合的UBZ结构域和PIP2结构域，但保留PIP1结构域，促进人类细胞中正常水平的跨损伤合成(TLS)，而不是Acis-synTT二聚体。在这里，我们确定了Polκ中的两个PIP结构域，并表明Tls在表达突变的Polκ的人成纤维细胞中正常存在，但这两个PIP结构域的突变失活使得Tls中的Polκ在胸腺嘧啶二醇损伤相反的TLS中不起作用。因此，在人类细胞中，PolDNA的两个PiP结构域在与这种κ损伤相反的TLS中冗余地发挥作用。然而，令人惊讶的是，尽管PIP1结构域的突变失活完全抑制了Polκ在增殖细胞核抗原、复制因子C和复制蛋白A存在下对DNA合成的刺激，但PIP2的突变对依赖于增殖细胞核抗原的DNA合成没有负面影响。这增加了POLPCNAPIP2作为κ结合域的激活在人类细胞的TLS过程中发生的可能性，并且这种激活涉及蛋白质-蛋白质相互作用和转录后修饰。
Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA‐binding motifs, PIP1 and PIP2, and that a C‐terminal deletion of Polη that lacks the ubiquitin‐binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite acis‐synTT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which thepip1orpip2mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA‐dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA‐binding domain occurs during TLS in human cells and that protein–protein interactions and post‐transcriptional modifications are involved in such activation.