Department of Chemistry and Biochemistry, Baylor University, Waco, TX, 76798, USA.
Department of Biology, New York University, New York, NY, 10003, USA.
DNA Repair (Amst). 2021 Nov;107:103194. doi: 10.1016/j.dnarep.2021.103194. Epub 2021 Jul 29.
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 能够识别多种 DNA 损伤，从而启动核苷酸切除修复（NER）。然而，不同损伤的 NER 倾向性差异很大，且修复抗性损伤具有持续性，因此具有高度致突变性。Rad4 通过解旋（“打开”）受损 DNA 位点来识别具有修复能力的损伤。当将 Rad4 连接到连续的 C/G（CCC/GGG）的正常 DNA 序列上以防止蛋白质扩散时，也会观察到这种“打开”。然而，尚不清楚这种连接促进的 DNA“打开”是否可以在任何 DNA 上发生，或者某些结构/序列是否会抵抗“打开”。在这里，我们报告说，即使与 Rad4 连接，含有交替 C/G（CGC/GCG）的 DNA 也无法被打开；相反，Rad4 以 180°反向方式结合，封闭了 DNA 末端。溶液中 DNA 构象的荧光寿命研究表明，CCC/GGG 表现出局部预熔解，而 CGC/GCG 则不存在。在 MD 模拟中，CGC/GCG 无法与 Rad4 结合以促进“打开”，而 CCC/GGG 则可以。总的来说，我们的研究说明了局部序列如何影响 Rad4/XPC 对 DNA 的识别，以及某些 DNA 位点如何即使在 Rad4 被固定在该位点的情况下也能抵抗“打开”。Rad4-DNA 识别中 CCC/GGG 和 CGC/GCG 序列之间的差异可能有助于解释各种基因组序列背景下的致突变性，从而解释损伤决定的突变热点和冷点。
