Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1061.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1061.
J Biol Chem. 2020 May 1;295(18):5918-5927. doi: 10.1074/jbc.RA120.012816. Epub 2020 Mar 13.
The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.
复制和跨损伤合成（TLS）DNA 聚合酶（Pols）的生化和结构分析揭示的作用机制在其细胞功能中得以保留。在这方面，DNA 聚合酶θ与其他 Pols 不同，因为纯化的 Polθ 以类似无碱基的模式在 1，-乙叉脱氧腺苷（ϵdA）的对面错误掺入一个 A，而 Polθ 在人细胞中主要进行无差错的 TLS。为了检验 Polθ 在人细胞中复制通过 ϵdA 时采用不同于纯化 Pol 的机制的假设，我们在此分析了高度保守的酪氨酸残基 Tyr-2387 和 Tyr-2391 在 Polθ 活性位点中的突变的影响。我们发现这些残基对于纯化 Pol 的 TLS 是必不可少的，但在人细胞中不需要，以及其他发现，为 Polθ 活性位点在人细胞中重新配置以稳定 ϵdA 处于 Hoogsteen 碱基配对与正确核苷酸的构象提供了强有力的证据。证据表明，一种 DNA 聚合酶可以在人细胞中与在纯化 Pol 中完全不同地配置其活性位点，为 DNA 聚合酶功能建立了一个新的范例。
