Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro

Rika Kusumoto , Chikahide Masutani , Kaoru Sugasawa , Shigenori Iwai , Marito Araki , Akio Uchida , Toshimi Mizukoshi , Fumio Hanaoka
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引用次数: 129

Abstract

The XPC–HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC–HR23B for defined lesions on DNA. Here we show that XPC–HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC–HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC–HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC–HR23B initiates global genomic NER. Although, XPC–HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC–HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC–HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC–HR23B but is not initiated by XPC–HR23B. Possible mechanisms will be discussed.

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体外全基因组核苷酸切除修复中损伤识别步骤的多样性
XPC-HR23B复合物是一种专门参与全局基因组核苷酸切除修复(NER)的哺乳动物因子,已被证明可以结合各种形式的受损DNA并在无细胞反应中启动DNA修复。为了更详细地表征该因子的结合特异性,我们开发了一种基于免疫沉淀的方法来评估XPC-HR23B对DNA上特定病变的相对亲和力。在这里,我们发现XPC-HR23B优先与紫外线诱导的(6-4)光产物(6-4PPs)以及胆固醇结合,但不与环丁烷嘧啶二聚体(CPD), 8-氧鸟嘌呤(8-氧鸟嘌呤),o6 -甲基鸟嘌呤(o6 -甲基鸟嘌呤)或单一错配结合。人全细胞提取物能以xpc - hr23b依赖的方式有效去除6-4PPs和胆固醇,但不能去除8-oxo-G、O6-Me-G或错配物。因此,XPC-HR23B对某些类型病变的结合特异性与人类细胞提取物切除这些病变的能力之间存在良好的相关性,支持XPC-HR23B启动全局基因组NER的模型。虽然XPC-HR23B并不优先结合CPDs,但在人全细胞提取物中CPDs的切除完全依赖于XPC-HR23B,这与在体内观察到的XP-C突变细胞中CPDs没有从全基因组中移除一致。这些结果表明,除了由XPC-HR23B启动的切除修复途径外,全球基因组NER还存在另一个子途径,该途径仍需要XPC-HR23B,但不是由XPC-HR23B启动的。将讨论可能的机制。
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