Molecular Pharmacology Department, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4057-62. doi: 10.1073/pnas.0909917107. Epub 2010 Feb 16.
Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5' extensions showed a high frequency of misrepair in Deltatdp1 cells. The joining errors in Deltatdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3' extensions or blunt ends did not show enhanced frequencies of errors, although Deltatdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3' nucleosidase activity regulates the processing of DNA ends by generating a 3' phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3' phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3' phosphate is a key step in Tdp1-mediated error prevention during NHEJ.
酪氨酸-DNA-磷酸二酯酶 1（Tdp1）可以使与 DNA 共价结合的肽断裂。我们评估了 Tdp1 在非同源末端连接（NHEJ）中的作用，发现具有 5'延伸的线性 DNA 分子在 Deltatdp1 细胞中显示出高频率的错误修复。Deltatdp1 细胞中的连接错误主要是 2-4 个核苷酸的插入。具有 3'延伸或平头末端的末端没有显示出增强的错误频率，尽管 Deltatdp1 细胞比 WT 细胞更有效地修复平头 DNA 末端。我们发现插入需要 Ku80 和 DNA 连接酶 IV，以及聚合酶 IV。我们的结果表明，酵母 Tdp1 是 NHEJ 途径的一个组成部分。我们建议 Tdp1p 3'核酸酶活性通过产生 3'磷酸来调节 DNA 末端的处理，从而限制聚合酶和其他酶在 DNA 末端的作用能力。为了支持这个模型，我们发现酵母 DNA 3'磷酸酶 Tpp1 的过表达也导致插入频率增加，这表明生成 3'磷酸是 Tdp1 在 NHEJ 过程中防止错误的关键步骤。
