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首页> 外文期刊>Molecular and Cellular Biology >HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.
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HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

机译：HHR23B是人类Rad23的同源物，可在体外核苷酸切除修复中刺激XPC蛋白。

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摘要

A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.
机译：先前已从HeLa细胞中纯化了一种特异性补充XP-C细胞提取物缺陷的蛋白质复合物，使其接近同质。该复合物由两个紧密相关的蛋白质组成：XPC基因产物和HHR23B，酿酒酵母修复基因产物Rad23的两个人同源物之一（Masutani等人，EMBO J.13：1831-1843，1994）。为了阐明这些蛋白质在“基因组整体”修复中的作用，我们在杆状病毒系统中表达了XPC蛋白质，并将其纯化至接近同质。重组人XPC（rhXPC）蛋白对单链DNA表现出高亲和力，并且无需额外添加重组HHR23B（rHHR23B）蛋白即可纠正XP-C全细胞提取物中的修复缺陷。但是，蛋白质印迹（免疫印迹）实验表明XP-C细胞提取物含有过量的内源性HHR23B蛋白，在添加rhXPC蛋白后可能会形成复合物。为了研究HHR23B的作用，我们对XP-C细胞提取物进行分级分离，并构建了既不存在内源XPC也不存在HHR23B蛋白的重构系统。在该测定系统中，单独使用rhXPC可以弱化修复缺陷，而在共添加rHHR23B蛋白后观察到了明显增强的纠正活性。发现了HHR23B刺激猿猴病毒40微染色体，裸质粒DNA，UV以及N-乙酰氧基-2-乙酰基芴诱导的DNA损伤，表明HHR23B在XPC中在基因组中起作用的一般作用-整体核苷酸切除修复子途径。

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