Binding of Escherichia coli DNA photolyase to UV-irradiated DNA.
Binding of Escherichia coli DNA photolyase to UV-irradiated DNA.
复制标题
大肠杆菌 DNA 光解酶与紫外线照射的 DNA 的结合。
DOI：
10.1021/bi00329a007
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Sancar,A
中科院分区：
文献类型：
--
作者：
Sancar,GB;Smith,FW;Sancar,A
大肠杆菌 DNA 光裂解酶是一种黄素蛋白，可催化 UV 照射下 DNA 中产生的嘧啶二聚体的光单体化。在体内，该酶通过两步机制发挥作用：它在不依赖于光的反应中与含有二聚体的 DNA 结合，并在暴露于 300-500 nm 光时破坏环丁烷环并与底物解离。使用纯化至同质的光裂合酶，我们在体外研究了反应的第一步，DNA 结合；通过硝化纤维素滤膜结合测定对酶-DNA复合物的形成进行定量。我们发现，无论 DNA 是超螺旋、开环还是线性形式，或者 DNA 是单链还是双链，该酶都能特异性结合紫外线照射的 DNA。结合反应在 NaCl 浓度为 125 mM、pH 7.5 时最佳。尽管光裂合酶被硝化纤维素过滤器以接近 100% 的效率保留，但单个酶-底物复合物的结合效率约为 0.34。通过将复合物暴露在溶液中或过滤器上的光活化光下，可以将其解离。 嘧啶二聚体是 DNA 中通过 254 nm 照射产生的主要光产物[参见 Patrick & Rahn (1976)]，并且是负责 UV 诱导突变和致死性的主要损伤[参见 Witkin (1976)]。在大肠杆菌以及其他生物体中，已经进化出许多酶来识别这些二聚体并通过各种机制将它们从 DNA 中去除（Lindahl，1982；Haseltine，
Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposureto 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although photolyase is retained by the nitrocellulose filterswith near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.^^ rimidine dimers are the major photoproducts produced in DNA by irradiation at 254 nm [see Patrick & Rahn (1976)] and are the primary lesions responsible for UV-induced mu-tagenesis and lethality [see Witkin (1976)]. In Escherichia coli as well as other organisms, a number of enzymes have evolved which recognize these dimers and remove them from DNA by various mechanisms (Lindahl, 1982; Haseltine,