Ma L, Siemssen E D, Noteborn H M, van der Eb A J
Laboratory for Molecular Carcinogenesis, Sylvius Laboratories, Leiden University, The Netherlands.
Nucleic Acids Res. 1994 Oct 11;22(20):4095-102. doi: 10.1093/nar/22.20.4095.
The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair.
XPB/ERCC3基因可纠正人类遗传性疾病B组着色性干皮病中的核苷酸切除修复缺陷，并编码基础转录因子BTF2/TFIIH的最大亚基。XPB/ERCC3蛋白的一级序列具有许多已知和推定的解旋酶或解旋酶相关蛋白中发现的七个解旋酶基序的特征。最近，已发现多蛋白BTF2/TFIIH复合物与DNA解旋酶活性有关。为了探索XPB/ERCC3的特性和功能，我们使用杆状病毒/昆虫细胞表达系统来生产重组蛋白。我们在此报告表达XPB/ERCC3的重组杆状病毒的构建和分析。XPB/ERCC3蛋白在杆状病毒感染的昆虫细胞中以相对较高的水平合成。虽然大多数XPB/ERCC3最终存在于昆虫细胞裂解物的不溶部分中，但一小部分重组蛋白以可溶形式存在，可在天然条件下纯化。我们发现纯化的XPB/ERCC3蛋白具有DNA解旋酶活性，这表明XPB/ERCC3可能在转录和核苷酸切除修复过程中作为DNA解旋酶在局部解开DNA模板方面发挥作用。
