The DNA helicase and adenosine triphosphatase activities of yeast Rad3 protein are inhibited by DNA damage. A potential mechanism for damage-specific recognition.
The DNA helicase and adenosine triphosphatase activities of yeast Rad3 protein are inhibited by DNA damage. A potential mechanism for damage-specific recognition.
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DOI：
10.1016/s0021-9258(18)48507-9
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发表时间：
1992-01
期刊：
影响因子：
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通讯作者：
Hanspeter Naegeli;L. Bardwell;E. Friedberg
中科院分区：
文献类型：
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作者：
Hanspeter Naegeli;L. Bardwell;E. Friedberg
从酵母中纯化的Rad3蛋白是一种单链DNA依赖的atp酶，也作为部分双链DNA的DNA解旋酶。在这项研究中，我们表明DNA解旋酶活性被抑制时，部分双工环状DNA底物暴露于紫外线（UV）辐射。DNA解旋酶活性的抑制对携带损伤的双链区域的特定链很敏感。如果单链环在退火到未辐照的寡核苷酸之前被辐照，则抑制作用保留，但如果紫外线辐照的寡核苷酸被退火到未辐照的环状单链DNA则不保留。紫外线照射单链DNA或脱氧核糖核苷酸均聚物也会抑制这些多核苷酸支持Rad3蛋白水解ATP的能力。紫外线辐射损伤明显阻断Rad3蛋白的易位，并导致Rad3蛋白-紫外线辐射DNA复合物的形成。因此，Rad3蛋白仍然被隔离在DNA上，可能是在碱基损伤的位置。Rad3蛋白对其易位链上DNA损伤的敏感性为核苷酸切除修复过程中的损伤识别提供了一种潜在的机制，并可能解释了酵母中损伤特异性DNA切口对Rad3蛋白的绝对需求。
Purified Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase and also acts as a DNA helicase on partially duplex DNA. In this study we show that the DNA helicase activity is inhibited when a partially duplex circular DNA substrate is exposed to ultraviolet (UV) radiation. Inhibition of DNA helicase activity is sensitive to the particular strand of the duplex region which carries the damage. Inhibition is retained if the single-stranded circle is irradiated prior to annealing to an unirradiated oligonucleotide, but not if a UV-irradiated oligonucleotide is annealed to unirradiated circular single-stranded DNA. UV irradiation of single-stranded DNA or deoxyribonucleotide homopolymers also inhibits the ability of these polynucleotides to support the hydrolysis of ATP by Rad3 protein. UV radiation damage apparently blocks translocation of Rad3 protein and results in the formation of stable Rad3 protein-UV-irradiated DNA complexes. As a consequence, Rad3 protein remains sequestered on DNA, presumably at sites of base damage. The sensitivity of Rad3 protein to the presence of DNA damage on the strand along which it translocates provides a potential mechanism for damage recognition during nucleotide excision repair and may explain the absolute requirement for Rad3 protein for damage-specific incision of DNA in yeast.