Squamous cell carcinoma (SCC) occurs frequently in the human Xeroderma Pigmentosum (XP) syndrome and is characterized by deficient UV-damage repair. SCC is the most common equine ocular cancer and the only associated genetic risk factor is a UV-damage repair protein. Specifically, a missense mutation in horse DDB2 (T338M) was strongly associated with both limbal SCC and third eyelid SCC in three breeds of horses (Halflinger, Belgian, and Rocky Mountain Horses) and was hypothesized to impair binding to UV-damaged DNA. Here, we investigate DDB2-T338M mutant’s capacity to recognize UV lesions in vitro and in vivo, together with human XP mutants DDB2-R273H and -K244E. We show that the recombinant DDB2-T338M assembles with DDB1, but fails to show any detectable binding to DNA substrates with or without UV lesions, due to a potential structural disruption of the rigid DNA recognition β-loop. Consistently, we demonstrate that the cellular DDB2-T338M is defective in its recruitment to focally radiated DNA damages, and in its access to chromatin. Thus, we provide direct functional evidence indicating the DDB2-T338M recapitulates molecular defects of human XP mutants, and is the causal loss-of-function allele that gives rise to equine ocular SCCs. Our findings shed new light on the mechanism of DNA recognition by UV-DDB and on the initiation of ocular malignancy.

鳞状细胞癌 (SCC) 经常发生在人类色素性干皮病 (XP) 综合征中，其特征是紫外线损伤修复不足。SCC 是最常见的马眼癌，唯一相关的遗传风险因素是紫外线损伤修复蛋白。具体而言，马 DDB2 (T338M) 的错义突变与三种马（哈弗林格马、比利时马和落基山马）的角膜缘 SCC 和第三眼睑 SCC 密切相关，并被假设会损害与紫外线损伤 DNA 的结合。在这里，我们研究了 DDB2-T338M 突变体在体外和体内识别紫外线损伤的能力，连同人类 XP 突变体 DDB2-R273H 和 -K244E。我们表明重组 DDB2-T338M 与 DDB1 组装在一起，但由于刚性 DNA 识别 β 环的潜在结构破坏，在有或没有紫外线损伤的情况下未显示与 DNA 底物的任何可检测结合。一贯地，我们证明细胞 DDB2-T338M 在其募集到局灶性辐射 DNA 损伤和获取染色质方面存在缺陷。因此，我们提供了直接的功能证据，表明 DDB2-T338M 概括了人类 XP 突变体的分子缺陷，并且是导致马眼部 SCC 的因果功能丧失等位基因。我们的研究结果为 UV-DDB 识别 DNA 的机制和眼部恶性肿瘤的发生提供了新的思路。