Caldecott K W, McKeown C K, Tucker J D, Ljungquist S, Thompson L H
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808.
Mol Cell Biol. 1994 Jan;14(1):68-76. doi: 10.1128/mcb.14.1.68-76.1994.
XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.
XRCC1是一种人类基因，它能完全校正中国仓鼠卵巢DNA修复突变体EM9，编码一种参与DNA单链断裂重新连接的蛋白质，这种单链断裂在经烷化剂或电离辐射处理后出现。在本研究中，构建了一个编码寡聚组氨酸标签XRCC1的cDNA小基因，以促进重组蛋白的亲和纯化。这个构建体命名为pcD2EHX，它完全校正了高姐妹染色单体交换的EM9表型，表明组氨酸标签对XRCC1活性无害。用pcD2EHX转染的EM9细胞提取物进行亲和层析，结果使组氨酸标签的XRCC1和DNA连接酶III活性共纯化。在用编码无标签XRCC1的cDNA小基因pcD2EX转染的EM9细胞提取物、野生型AA8细胞提取物或未转染的EM9细胞提取物进行亲和层析时，XRCC1或DNA连接酶III活性均未被纯化。DNA连接酶III活性与组氨酸标签的XRCC1共纯化表明这两种蛋白质在细胞中以复合物形式存在。此外，EM9细胞中DNA连接酶III活性水平低于AA8细胞，在用pcD2EHX或pcD2EX转染的EM9细胞中恢复到正常水平。这些发现表明，正常水平的DNA连接酶III活性需要XRCC1，并且暗示了这种DNA连接酶在哺乳动物细胞DNA碱基切除修复中的主要作用。
