Yamada Daichi, Yamamoto Junpei, Getzoff Elizabeth D, Iwata Tatsuya, Kandori Hideki
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Graduate School of Science, University of Hyogo, Hyogo 678-1297, Japan.
Biochemistry. 2021 Nov 2;60(43):3253-3261. doi: 10.1021/acs.biochem.1c00413. Epub 2021 Oct 17.
Photolyases (PHRs) repair ultraviolet (UV)-induced DNA photoproducts into normal bases. In this study, we measured the conformational changes upon photoactivation and photorepair processes of a PHR and its specific substrates, (6-4)PHR and a pyrimidine(6-4)pyrimidone photoproduct ((6-4)PP), by light-induced difference Fourier transform infrared (FT-IR) spectroscopy. The single-stranded DNA with (6-4)PP (ss(6-4)PP) was used as a substrate and the resultant FT-IR spectra were compared with the previous results on double-stranded DNA with (6-4)PP (ds(6-4)PP). In the excess amount of substrate to the enzyme, different ss(6-4)PP photorepair FT-IR signals were obtained in an illumination time-dependent manner. As reported for ds(6-4)PP, the early stages of the photoreaction involve the changes in the ss(6-4)PP only, while the late stages of the reaction involve the ss(6-4)PP repair-associated changes and dissociation from (6-4)PHR. From these spectra, difference spectra originating from the binding/dissociation spectrum were extracted. The signals of the C═O stretches of (6-4)PP and repaired thymines in the single- and double-stranded DNA were tentatively assigned. The C═O stretches of (6-4)PP were observed at frequencies that reflect single- and double-stranded DNA environments in aqueous solution, reflecting the different hydrogen-bonding environments. The conformational changes of PHR upon binding of ss(6-4)PP and ds(6-4)PP were similar, suggesting that the conformational change is limited to the (6-4)PP binding pocket region. We interpreted that ds(6-4)PP may be bound together without any special mechanism for flipping out.
光解酶（PHRs）可将紫外线（UV）诱导产生的DNA光产物修复为正常碱基。在本研究中，我们通过光诱导差示傅里叶变换红外（FT-IR）光谱法，测量了一种光解酶及其特定底物（6-4）光解酶和嘧啶（6-4）嘧啶酮光产物（（6-4）PP）在光激活和光修复过程中的构象变化。以含有（6-4）PP的单链DNA（ss(6-4)PP）作为底物，并将所得的FT-IR光谱与先前关于含有（6-4）PP的双链DNA（ds(6-4)PP）的结果进行比较。在底物相对于酶过量的情况下，以光照时间依赖的方式获得了不同的ss(6-4)PP光修复FT-IR信号。正如ds(6-4)PP的报道，光反应的早期阶段仅涉及ss(6-4)PP的变化，而反应的后期阶段涉及与ss(6-4)PP修复相关的变化以及与（6-4）光解酶的解离。从这些光谱中，提取了源自结合/解离光谱的差示光谱。初步确定了单链和双链DNA中（6-4）PP和修复后的胸腺嘧啶的C═O伸缩振动信号。在反映水溶液中单链和双链DNA环境的频率处观察到（6-4）PP的C═O伸缩振动，这反映了不同的氢键环境。ss(6-4)PP和ds(6-4)PP结合时光解酶的构象变化相似，表明构象变化仅限于（6-4）PP结合口袋区域。我们推断ds(6-4)PP可能无需任何特殊的翻转机制就能结合在一起。
