Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Biology, New York University, New York, NY 10003, USA.
Nucleic Acids Res. 2018 Jul 27;46(13):6455-6469. doi: 10.1093/nar/gky444.
5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA-protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130-14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and -1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.
5- 甲酰胞嘧啶（5fC）是一种通过 TET 蛋白介导的 5-甲基-dC 氧化作用引入的表观遗传 DNA 修饰。我们最近报道，5fC 可与活细胞中的组蛋白蛋白形成可逆的 DNA-蛋白缀合物（DPC）（Ji 等人，2017 年，Angew. Chem. Int. Ed.，56：14130-14134）。现在，我们研究了 5fC 介导的 DPC 对 DNA 复制的影响。在存在人类跨损伤合成 DNA 聚合酶 η 和 κ 的情况下，将含有特定位置的 DPC 的合成 DNA 双链体进行引物延伸实验，其中 5fC 与含有赖氨酸的蛋白质和肽之间存在 DPC。我们发现，包含组蛋白 H2A 或 H4 的 DPC 完全抑制了 DNA 复制，但当蛋白质进行蛋白水解消化时，复制被阻止。两种聚合酶以易错的方式绕过与 11- mer 或 31- mer 肽的交联，诱导靶向 C→T 转换和-1 缺失。当含有 5fC- 肽交联的质粒在人胚肾（HEK）293T 细胞中复制时，观察到类似类型的突变。结合人聚合酶 η 和 κ 的 11- mer 肽-dC 交联的分子模拟表明，肽很好地适合于 DNA 大沟侧，并且修饰的 dC 通过在聚合酶活性位点中的摆动碱基配对与进入的 dATP 形成稳定的错配。
