Huang J C, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
J Biol Chem. 1994 Jul 22;269(29):19034-40.
Human cells remove bulky adducts from DNA by excising single-stranded fragments 27-29 nucleotides in length by an enzyme system consisting of at least 14 polypeptides. All of the previous work on characterizing the excision reaction was conducted with plasmids 3 or 8 kilobases in length. To determine if the size and tertiary structure of DNA play a role in the excision reaction and to find out if large DNA fragments are necessary to contact all of the subunits of the excinuclease, we performed experiments with circular DNA and with linear DNA fragments of various sizes. We found that the human excinuclease is capable of removing DNA adducts from linear and covalently closed circular DNAs with about the same efficiency. Furthermore, we found that the excinuclease can remove a thymine dimer or a psoralen-thymine monoadduct from linear fragments provided that the distance between the lesion and the 5'-terminus of the damaged strand is > or = 60 nucleotides and the distance between the lesion and the 3'-terminus is > or = 44 nucleotides. Thus, the minimum size substrate for human excinuclease is approximately 100 base pairs in length.
人类细胞通过一种由至少14种多肽组成的酶系统切除长度为27 - 29个核苷酸的单链片段，从而从DNA中去除大分子加合物。此前所有关于表征切除反应的工作都是使用长度为3或8千碱基的质粒进行的。为了确定DNA的大小和三级结构是否在切除反应中起作用，以及找出大的DNA片段是否是接触核酸外切酶所有亚基所必需的，我们用环状DNA和各种大小的线性DNA片段进行了实验。我们发现人类核酸外切酶能够以大致相同的效率从线性和共价闭合环状DNA中去除DNA加合物。此外，我们发现只要损伤与受损链5'端之间的距离≥60个核苷酸，且损伤与3'端之间的距离≥44个核苷酸，核酸外切酶就能从线性片段中去除胸腺嘧啶二聚体或补骨脂素 - 胸腺嘧啶单加合物。因此，人类核酸外切酶的最小底物长度约为100个碱基对。
