School of Public Health, Xinjiang Medical University , Urumqi 830011, China.
Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University , Chengdu 610041, China.
Chem Res Toxicol. 2018 Jan 16;31(1):58-65. doi: 10.1021/acs.chemrestox.7b00287. Epub 2017 Dec 11.
The abasic site is one the most common DNA lesions formed in cells; it induces a severe blockage of DNA replication and is highly mutagenic. We continue to use Gp90 exo, the sole DNA polymerase from Pseudomonas aeruginosa phage PaP1, to study DNA replication upon encountering an abasic site lesion. Gp90 exo can incorporate dNTPs opposite the abasic site, but extension past this site is extremely slow. Among the four dNTPs, dATP is preferentially incorporated opposite the abasic site, consistent with the A-rule. The incorporation is independent of the identity of the nucleotide 5' of the abasic site. The incorporation of dATP opposite the abasic site occurs by direct incorporation of dNTP opposite the abasic site without a -1 frameshift deletion. Extension from an A:abasic site pair by Gp90 exo is slightly unfavorable relative to those from other abasic site pairs. Incorporation of dATP opposite the abasic site is preferential and shows a biphasic shape, indicating that this incorporation is much faster than the subsequent dissociation of the polymerase from DNA. The template sequence does not affect the dATP incorporation priority, burst amplitude, burst rate, or dATP dissociation constant. Surface plasmon resonance shows that the presence of an abasic site in the template weakens the binding affinity of Gp90 exo to DNA in a binary or ternary complex in the presence of any one kind of dNTP. This study reveals that Gp90 exo preferentially inserts A opposite an abasic site via the A-rule, like other DNA polymerases (e.g., Pol θ, KlenTaq, KF exo, Pols α, δ/PCNA, and Thermococcus litoralis Pol Vent (exo)), providing further insight into DNA replication mediated by P. aeruginosa phage PaP1 upon encountering an abasic site lesion.
碱基位点是细胞中形成的最常见的 DNA 损伤之一；它会严重阻碍 DNA 复制，并且具有高度的突变性。我们继续使用 Gp90 exo，即来自铜绿假单胞菌噬菌体 PaP1 的唯一 DNA 聚合酶，来研究遇到碱基缺失损伤时的 DNA 复制。Gp90 exo 可以在碱基缺失位点的对面掺入 dNTP，但延伸超过这个位点非常缓慢。在四种 dNTP 中，dATP 优先掺入碱基缺失位点的对面，符合 A 规则。这种掺入与碱基缺失位点 5'核苷酸的身份无关。dATP 直接掺入碱基缺失位点对面而不发生 -1 移框缺失，从而发生碱基缺失位点对面的 dATP 掺入。与其他碱基缺失对相比，Gp90 exo 从 A:碱基缺失对延伸稍微不利。Gp90 exo 优先在碱基缺失位点对面掺入 dATP，并表现出双相形状，表明这种掺入比随后聚合酶从 DNA 上的解离快得多。模板序列不影响 dATP 掺入优先级、爆发幅度、爆发率或 dATP 解离常数。表面等离子体共振显示，在存在任何一种 dNTP 的情况下，模板中碱基缺失位点的存在会削弱 Gp90 exo 在二元或三元复合物中与 DNA 的结合亲和力。这项研究表明，Gp90 exo 通过 A 规则优先在碱基缺失位点的对面插入 A，就像其他 DNA 聚合酶（例如 Pol θ、KlenTaq、KF exo、Pols α、δ/PCNA 和 Thermococcus litoralis Pol Vent(exo)）一样，进一步深入了解了铜绿假单胞菌噬菌体 PaP1 在遇到碱基缺失损伤时介导的 DNA 复制。
