Molecular Analysis of DNA Repair Defect and Nnurological Abnormalities in Group a Xeroderma Pigmentosum

我们克隆了一个小鼠DNA切除修复基因，它补充了A组着色性干皮病（XP）的缺陷，并命名为XPAC基因。我们还克隆了一个编码273个氨基酸残基的人XPAC cDNA和一个约25 kb长的人XPAC基因，该基因分为六个外显子。XPAC cDNA的表达赋予了几个A组XP细胞系的UV抗性，但对其他XP组的线。几乎所有检测的A组XP株系均显示XPAC mRNA异常或缺失。这些结果表明，有缺陷的XPAC基因导致A组XP。人XPAC cDNA编码相对分子质量为31 K的亲水性蛋白。XPC蛋白含有C4型锌指基序，表明它直接与DNA相互作用。重组融合蛋白在大肠杆菌中表达。大肠杆菌T7表达系统和显微注射重组xpac蛋白可恢复UV或4 NQO诱导的A组XP细胞的程序外DNA合成。用免疫荧光法检测抗XPAC蛋白的多克隆抗体 ...更多信息 将重组xpac蛋白导入家兔体内。用该抗体免疫沉淀细胞粗提物，SDS-PAGE检测正常人细胞中约40 K和38 K的xpac蛋白，但在A组XP细胞中未检测到。免疫荧光研究表明，xpac蛋白定位于细胞核，从而探讨了A组XP的分子基础。我们在日本A组XP患者中发现了三种不同类型的XPAC基因突变。一个是内含子3的3'剪接受体位点的G -> C颠换，其将强制性AG受体二核苷酸改变为AC，产生两种异常剪接的mRNA形式。这种单碱基取代为AlwNI限制性内切核酸酶产生新的切割位点。AlwNI RFLP分析显示，这种突变在日本A组XP患者中的频率很高。第二种是第6外显子C → T转换，改变Arg密码子（CGA）为无义密码子（TGA）。一名日本A组XP患者表现出轻微的皮肤症状，没有皮肤肿瘤或神经异常，是该突变的纯合子。第三种是第三外显子的T → A颠换，将Tyr密码子（达特）变为无义密码子（TAA）。在21例无关的日本组AXP患者中，2例临床症状严重的患者具有该突变等位基因。少

We cloned a mouse DNA excision repair gene which complements the defect of group A xeroderma pigmentosum (XP) and named it the XPAC gene. We also cloned a human XPAC cDNA that encodes 273 amino acid residues and a human XPAC gene that is about 25kb long and is split into six exons. Expression of XPAC cDNA conferred UV-resistance on several group A XP cell lines, but on lines of other XP groups. Almost all group A XP lines tested showed abnormality or absence of XPAC mRNA. These results indicate that a defective XPAC gene causes group A XP. Human XPAC cDNA encodes a hydrophilic protein of relative molecular mass 31K. The XPC protein contains C4 type zinc-finger motif, indicating that it interacts directly with DNA. The recombinant fused XPAC protein was produced in E. coli by T7 expression system and microinjection of the recombinant xpac protein restored UV or 4NQO-induced unscheduled DNA synthesis in group A XP cells. The polylonal anti xpac protein antibody was elicited by injecting … More the recombinant xpac protein into rabbit. Immunoprecipitation of crude cell extract with this antibody and SDS-PAGE detected about 40K and 38K xpac protein in normal human cells, but not in group A XP cells. Immunofluorescence study revealed that xpac protein localized in the cell nucleus.The molecular basis of group A XP was then investigated. We found three different kinds of mutations of XPAC gene in Japanese group A XP patients. One was a G -> C transversion at the 3' splice acceptor site of intron 3, which altered the obligatory AG acceptor dinucleotides to AC, creating two abnormally spliced mRNA forms. This single base substitution creates a new cleavage site for AlwNI restriction endonuclease. Analysis of AlwNI RFLP showed a high frequency of this mutation in Japanese group A XP patients. Second one was C -> T transition in exon 6 altering Arg codon (CGA) to a nonsense codon (TGA). One of the Japanese group A XP patients who showed mild skin symptoms with no skin tumors or neurological abnormalities was a homozygote for this mutation. The third one was a T -> A transversion in exon 3 altering Tyr codon (TAT) to a nonsense codon (TAA). Of 21 unrelated Japanese group AXP patients, 2 with severe clinical symptoms had this mutant alleles. Less