Svedruzić Zeljko M, Wang Chenbo, Kosmoski Joseph V, Smerdon Michael J
Department of Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
J Biol Chem. 2005 Dec 2;280(48):40051-7. doi: 10.1074/jbc.M509478200. Epub 2005 Oct 6.
Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30 degrees bending and 9 degrees unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.
环丁烷胸腺嘧啶二聚体（CTDs）是紫外线辐射诱导产生的最常见的DNA损伤，会导致DNA螺旋弯曲30度并解旋9度。我们在由强核小体定位序列包围的短序列内制备了位点特异性CTDs。通过羟基自由基足迹法分析了组蛋白表面二分体中心附近螺旋一圈上CTDs的旋转设置。令人惊讶的是，螺旋一圈上CTDs的位置并不影响核小体表面DNA的旋转设置。凝胶迁移分析表明，当一个CTD背离或朝向组蛋白表面时，分别会使组蛋白-DNA相互作用的稳定性降低0.6或1.1 kJ/mol。因此，对于一个埋藏的CTD，0.5 kJ/mol的能量损失不足以改变具有强烈旋转偏好的序列的旋转设置。使用非洲爪蟾卵母细胞核提取物研究了旋转设置对核苷酸切除修复（NER）去除CTD的影响。在核小体中，NER速率仅低2至3倍，当CTD背离或朝向组蛋白表面时，NER速率仅变化1.5倍。因此，在非洲爪蟾核提取物中，核小体上CTDs的旋转方向对修复速率的影响出奇地小。这些结果表明，核小体动力学和/或染色质重塑可能有助于NER接触核小体中的DNA损伤。
