Evans E, Fellows J, Coffer A, Wood R D
Imperial Cancer Research Fund, Clare Hall Laboratories, London, UK.
EMBO J. 1997 Feb 3;16(3):625-38. doi: 10.1093/emboj/16.3.625.
Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.
人类XPG核酸酶在DNA的核苷酸切除修复过程中进行3'端切割。该酶特异性地在未配对DNA与双链区域的交界处附近切割模型DNA气泡结构。然而，目前尚不清楚未配对结构是否是实际DNA修复过程中的一个中间体。我们在此发现，XPG需要打开>5个碱基对才能有效切割。为了寻求在体外核苷酸切除修复反应过程中DNA损伤周围形成开放结构的直接证据，我们对携带独特位置顺铂加合物的受损DNA分子进行了高锰酸钾足迹实验。观察到一个延伸约25个核苷酸的解旋开放复合物，其延伸至5'和3'切割位点的位置，并且依赖于XPA蛋白和ATP。修复过程中的开放发生在XPG进行链切割之前。
