School of Biological Engineering , Dalian Polytechnic University , Dalian , 116034 , China.
Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health and West China Fourth Hospital , Sichuan University , Chengdu , China.
Chem Res Toxicol. 2019 May 20;32(5):840-849. doi: 10.1021/acs.chemrestox.8b00348. Epub 2019 Apr 12.
N-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo. The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency ( k/ K) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate ( k), and Hyp increased the dissociation constant ( K). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.
N6- 甲基腺嘌呤（6mA）是一种新发现的表观遗传修饰，在调控多种生物学过程中发挥重要作用。然而，6mA 对 DNA 复制的影响尚未得到充分研究。在这项工作中，我们研究了 6mA 如何通过铜绿假单胞菌噬菌体 PaP1 的 DNA 聚合酶（gp90 exo）影响 DNA 复制。6mA 及其中间产物次黄嘌呤（Hyp）的存在抑制了 gp90 exo 的 DNA 复制。6mA 将 dTTP 的掺入效率降低了 10 倍，并将下一个碱基延伸效率降低了 100 倍。不同的是，dCTP 在四种 dNTP 中优先掺入 Hyp 对面。gp90 exo 将延伸优先级降低到 6mA:T 对之外，而不是 6mA:C 错配，并且更喜欢在 Hyp:C 对面延伸，而不是 Hyp:T 对面。dTTP 对 6mA 和 dCTP 对 Hyp 的掺入均显示出快速爆发阶段。与未修饰的 A 相比，6mA 的爆发率和爆发幅度均降低。此外，与 dTTP 对 A 的掺入相比，6mA 对面的 dTTP 掺入和 Hyp 对面的 dCTP 掺入的总掺入效率（k/K）降低。6mA 降低了掺入速率（k），Hyp 增加了解离常数（K）。然而，模板上的 6mA 或 Hyp 并不影响 DNA 聚合酶在二聚体或三聚体复合物中与 DNA 的结合。这项工作为 PaP1 中 6mA 的表观遗传修饰对 DNA 复制的抑制作用提供了新的见解。
