The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage
The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage
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DOI：
10.1093/emboj/16.4.889
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发表时间：
1997-02-17
期刊：
影响因子：
11.4
通讯作者：
Rupp, WD
中科院分区：
文献类型：
--
作者：
Gordienko, I;Rupp, WD
大肠杆菌Uvr核苷酸切除修复蛋白对具有不同化学结构的各种病变的识别以及UvrAB复合物中存在解旋酶活性（其可以置换退火至单链DNA的短寡核苷酸）导致了一种模型，其中该活性将UvrAB沿着未受损DNA移动至受损位点，在该位点处病变阻断进一步易位，并形成蛋白质-DNA切割前复合物。为了评估这种损伤识别机制，我们构建了不同长度的寡核苷酸退火到单链DNA环的基板，并放置一个单一的2-（乙酰氨基）芴（AAF）损伤的寡核苷酸或环上。对于没有损伤的底物，UvrAB复合物有效地置换了22-mer而不是27-mer或更长的片段。寡核苷酸上AAF的存在显著增加了27聚体的释放，但30或更长的寡聚体未分离。将病变部位置于环形股线上并不能阻止碎片的释放。相反，UvrAB的释放活性受到刺激，并且还取决于退火寡核苷酸的长度。这些观察结果不同意依赖于解旋酶驱动的易位的损伤识别机制的预测。最有可能的是，UvrAB的链分离活性是在受损部位形成DNA-蛋白质切割前复合物期间发生的局部变化的结果，而不是由于蛋白质沿着未受损DNA移位以定位病变。
The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.