Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Department of Biology, New York University, New York, New York 10003-6688, United States.
Biochemistry. 2021 Jun 15;60(23):1797-1807. doi: 10.1021/acs.biochem.1c00130. Epub 2021 Jun 3.
DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.
DNA-蛋白质交联（DPC）是一种异常庞大的 DNA 损伤，当细胞蛋白在暴露于紫外线、自由基、醛和过渡金属后被困在 DNA 上时就会形成。DPC 也可以在自然发生的表观遗传标记[5-甲酰胞嘧啶（5fC）]与组蛋白的赖氨酸和精氨酸残基反应形成席夫碱缀合物时内源性形成。我们之前的研究表明，DPC 抑制 DNA 复制和转录，但可以通过蛋白水解切割产生较小的 DNA-肽缀合物。我们已经表明，在 CXA 序列（X = DpC）内的 5fC 缀合的 DNA-肽交联（DpC）可以被人类跨损伤合成（TLS）聚合酶 η 和 κ 以易错的方式绕过。然而，局部核苷酸序列背景可以通过影响 DNA 模板、聚合酶和进入的 dNTP 之间的三元复合物的几何形状对大体积损伤的复制绕过产生强烈影响。在这项工作中，我们研究了在人类 TLS 聚合酶 η 的存在下，放置在七个不同序列背景（CXC、CXG、CXT、CXA、AXA、GXA 和 TXA）中的 5fC-DNA-11 肽交联的聚合酶绕过。通过凝胶电泳分析引物延伸产物，并研究了在不同碱基序列背景下 dAMP 错配掺入 DpC 损伤的稳态动力学。我们的结果表明，近邻碱基同一性对聚合酶 η 在不存在和存在 DpC 损伤时的活性有强烈影响。分子动力学模拟用于从结构上解释实验结果。我们的结果表明，在存在和不存在 DpC 损伤的情况下，局部 DNA 序列可能在促进与 TLS 相关的突变热点方面发挥作用。
