Petruseva Irina, Naumenko Natalia, Kuper Jochen, Anarbaev Rashid, Kappenberger Jeannette, Kisker Caroline, Lavrik Olga
Laboratory of Bioorganic Chemistry of Enzymes, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Wuerzburg, Wuerzburg, Germany.
Front Cell Dev Biol. 2021 Mar 11;9:617160. doi: 10.3389/fcell.2021.617160. eCollection 2021.
The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.
通过核苷酸切除修复（NER）系统成功消除大量DNA损伤在很大程度上取决于损伤识别步骤。这一步骤包括对损伤的初步识别和验证。TFIIH解旋酶XPD在NER的验证步骤中起关键作用。迄今为止，损伤验证机制尚未得到充分理解，需要进一步详细研究。本研究是对ctXPD以及ctXPD-ctp44与模型DNA相互作用的系统研究，这些模型DNA含有结构不同的大量损伤，且具有先前估计的NER修复效率。我们利用ATP酶和DNA结合研究来评估ctXPD与受损DNA的相互作用。对ctXPD-ctp44与含有荧光和光活化损伤的DNA结合的分析结果表明，XPD对含有大量损伤的DNA的亲和力与NER系统消除损伤的能力之间存在关联。ctXPD与含有可修复和不可修复光活化损伤的DNA探针的光交联揭示了在有和没有ctp44的情况下DNA相互作用效率的差异。总体而言，所获得的结果表明ctXPD-ctp44与损伤相互作用的能力，并表明ctp44亚基在验证过程中起重要作用。
