Yockey Oliver P, Jha Vikash, Ghodke Pratibha P, Xu Tianzuo, Xu Wenyan, Ling Hong, Pradeepkumar P I, Zhao Linlin
Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario , London, Ontario N6A 5C1, Canada.
Department of Chemistry, Indian Institute of Technology Bombay , Mumbai 400076, India.
Chem Res Toxicol. 2017 Nov 20;30(11):2023-2032. doi: 10.1021/acs.chemrestox.7b00227. Epub 2017 Oct 23.
DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N-2'-deoxyguanosine (N-dG) and N-2'-deoxyadenosine (N-dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N-dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.
DNA损伤影响遗传信息流，对人类疾病和衰老具有重要意义。卢西丁-3-O-樱草糖苷（LuP）是茜草根中存在的一种蒽醌衍生物，曾被用作着色剂和食品添加剂。LuP可通过代谢转化为具有基因毒性的化合物卢西丁，随后形成卢西丁特异性的N-2'-脱氧鸟苷（N-dG）和N-2'-脱氧腺苷（N-dA）DNA加合物。卢西丁在啮齿动物中具有致突变性和致癌性，但在人类中的致癌风险较低。为了解卢西丁在人类中低致癌性的分子机制，我们使用含有卢西丁-N-dG DNA加合物结构类似物（LdG）的位点特异性修饰寡脱氧核苷酸进行了DNA复制测定，并确定了与含LdG的DNA和进入的核苷酸形成复合物的DNA聚合酶（pol）κ的晶体结构。我们研究了四种人类pol（pol η、pol ι、pol κ和Rev1）在与LdG进行DNA复制过程中的效率和准确性；这些pol是跨损伤DNA合成的关键参与者。我们的结果表明，pol κ能够高效且准确地复制通过LdG加合物，而pol η、pol ι进行的DNA复制在不同程度上受到损害。Rev1保留了在损伤位点掺入dCTP的能力，尽管效率有所降低。pol κ的两个三元晶体结构表明，在插入和损伤后延伸步骤中，LdG加合物在酶活性位点被pol κ容纳。pol κ独特的开放活性位点允许加合的DNA采用标准B型以进行准确的DNA复制。总体而言，这些生化和结构数据为卢西丁在人类中低致癌风险提供了机制性见解。
