Codominance associated with overexpression of certain XPD mutations

Saloumeh Kadkhodayan , Frédéric Coin , Edmund P. Salazar , James W. George , Jean-Marc Egly , Larry H. Thompson
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引用次数: 4

Abstract

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive “repair” that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

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共显性与某些XPD突变的过表达有关
XPD基因突变与三种复杂的临床表型相关,即色素干皮病(XP)、XP合并Cockayne综合征(XP- cs)和毛硫营养不良(TTD)。XP是由核苷酸切除修复(NER)缺陷引起的,这会导致皮肤癌的高风险。TTD的特点是严重的发育和神经缺陷,以脆性头发和鳞状皮肤为特征,有时有缺陷的NER。我们使用CHO细胞作为一个系统来研究特定突变如何改变XPD蛋白的显性/隐性行为。之前,我们在两种高度紫外线敏感的仓鼠细胞系中发现了T46I和R75W突变,据报道,这两种细胞系具有高水平的计划外DNA合成。在这里,我们报告这些突变体在细胞提取切除实验中大大降低了XPD解旋酶活性和完全缺陷的NER。我们得出结论,在这些突变体中看到的计划外DNA合成是由失败的“修复”引起的,这对细胞存活没有贡献。这些突变以及K48R规范解旋酶结构域突变在野生型CHO细胞中过表达时均产生共显性阴性表型。常见的xp特异性R683W突变在过表达时也表现为共显性,这与这种突变可能主要影响蛋白质的酶活性而不是损害蛋白质相互作用的观点是一致的,这可能是TTD的基础。在TTD中唯一发现的c端突变(R722W)过度表达,但其水平不足以严格检测共显性表型。突变型XPD等位基因的过表达可能为在其他细胞系中产生NER缺陷提供了一种简单的方法。
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