Department of Chemistry and Chemical Biology, Center for Interdisciplinary Research on Complex Systems, Northeastern University, 360 Huntington Ave., 102 Hurtig Hall, Boston, MA 02115, USA.
J Bacteriol. 2011 Oct;193(19):5400-11. doi: 10.1128/JB.05301-11. Epub 2011 Jul 22.
DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD', form the specialized E. coli Y-family DNA polymerase UmuD'2C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.
DNA 经常受到化学物质和环境诱变剂的影响，导致复制停滞的损伤。为了应对 DNA 损伤和其他应激，大肠杆菌利用 SOS 反应，调节至少 57 个基因的表达，包括 umuDC。umuDC 的基因产物，umuC 和 UmuD 的切割形式，umuD'，形成了专门的大肠杆菌 Y 家族 DNA 聚合酶 umuD'2C，或聚合酶 V（Pol V）。Y 家族 DNA 聚合酶的特点是能够在称为跨损伤合成（TLS）的过程中复制受损 DNA，并且在未受损的 DNA 模板上具有低保真度。Y 家族聚合酶对不同类型的 DNA 损伤表现出不同的特异性。Pol V 进行 TLS 以绕过碱基缺失和紫外线辐射产生的胸腺嘧啶-胸腺嘧啶二聚体。通过丙氨酸扫描诱变，我们通过检测单个丙氨酸变体在紫外线诱导突变中的功能以及它们赋予对紫外线辐射抗性的能力，探测了由残基 31 到 38 和 50 到 54 组成的两个活性位点环在 Pol V 活性中的作用。我们发现，环 1 的 N 端残基 N32、N33 和 D34 的突变赋予了对紫外线辐射和 4-硝基喹啉-N-氧化物的超敏性，并显著降低了 Pol V 依赖性紫外线诱导突变。此外，突变 32、33 或 34 会降低 Pol V 依赖性抑制重组，表明这些突变可能破坏了 UmuC 与 RecA 的相互作用，这也可能导致表达这些变体的细胞对紫外线的超敏性。
