Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, USA.
Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.
Environ Mol Mutagen. 2024 Apr;65 Suppl 1(Suppl 1):25-39. doi: 10.1002/em.22570. Epub 2023 Sep 1.
In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~two-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.
针对氧化损伤，碱基切除修复（BER）酶使 VEGF 启动子的结构平衡在 B 型和 G4 DNA 构象之间发生波动，从而导致基因表达的表观遗传样修饰。然而，其机制细节仍然很神秘，包括 BER 酶在受损 G4 启动子上的活性和协调。为了解决这个问题，我们通过使用预稳态动力学测定法和体外偶联 BER 测定法来研究每个 BER 因子在 VEGF 启动子 G4 DNA 底物上进行其修复活性的能力。OGG1 能够在双链 VEGF 启动子 G4 DNA 底物上启动 BER。此外，预稳态动力学表明，与 B 型 DNA 相比，APE1 在 G4 上的修复活性降低了约两倍，这是由于产物释放较慢而不是链切割效率低下所致。有趣的是，与在 B 型 DNA 上的单个插入相比，Pol β 通过链置换 DNA 合成在 G4 底物上进行多次插入。多次插入抑制 Pol β 产物的连接，因此通过典型的短补丁 BER 无法完成 VEGF G4 启动子底物上的 BER。相反，修复需要 FEN1 的长补丁 BER 侧翼内切酶活性，以响应 Pol β 进行的多次插入，然后再进行连接。由于 BER 蛋白及其修复活性是 VEGF 转录增强对 G4 VEGF 启动子氧化 DNA 损伤的关键部分，因此，这里报告的关于该启动子中 BER 活性的新见解与理解转录调控机制有关。
