Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding (‘opening’) the damaged DNA site. Such ‘opening’ is also observed on a normal DNA sequence containing consecutive C/G’s (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA ‘opening’ could occur on any DNA or if certain structures/sequences would resist being ‘opened’. Here, we report that DNA containing alternating C/G’s (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote ‘opening’ contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being ‘opened’ even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion’s mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

Rad4/XPC 识别不同的 DNA 损伤以启动核苷酸切除修复 (NER)。然而，病变之间的 NER 倾向差异很大，并且修复抵抗性病变是持续存在的，因此具有高度诱变性。 Rad4 通过解开（“打开”）受损的 DNA 位点来识别修复能力强的损伤。当连接到 Rad4 以防止蛋白质扩散时，在包含连续 C/G (CCC/GGG) 的正常 DNA 序列上也观察到这种“打开”。然而，尚不清楚这种束缚促进的 DNA“打开”是否会发生在任何 DNA 上，或者某些结构/序列是否会抵抗“打开”。在这里，我们报道了含有交替 C/G (CGC/GCG) 的 DNA 即使在束缚时也无法打开；相反，Rad4 以 180° 反转的方式结合，给 DNA 末端加帽。溶液中 DNA 构象的荧光寿命研究表明，CCC/GGG 表现出 CGC/GCG 中不存在的局部预熔化。在MD模拟中，与CCC/GGG相反，CGC/GCG未能利用Rad4来促进“开放”。总而言之，我们的研究说明了局部序列如何影响 Rad4/XPC 的 DNA 识别，以及某些 DNA 位点如何抵抗“打开”，即使 Rad4 无限期地保留在该位点也是如此。 Rad4-DNA 识别中 CCC/GGG 和 CGC/GCG 序列之间的对比可能有助于破译病变在各种基因组序列背景下的致突变性，以解释病变决定的突变热点和冷点。