Hwang Hanshin, Taylor John-Stephen
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Biochemistry. 2004 Nov 23;43(46):14612-23. doi: 10.1021/bi0489558.
The Y family DNA polymerase yeast pol eta inserts pyrene deoxyribose monophosphate (dPMP) in preference to A opposite an abasic site, the 3'-T of a thymine dimer, and a normal T with almost equal efficiency. In contrast, pol A family polymerases such as Klenow fragment and T7 DNA polymerase only insert dPMP efficiently opposite an abasic site and the 3'-T of a thymine dimer but not opposite undamaged DNA. Pyrene nucleotide is also an efficient chain-terminating inhibitor of DNA synthesis by pol eta but not by Klenow fragment or T7 DNA polymerase. To better understand the origin of the efficiency and sequence specificity of dPMP insertion by pol eta, the kinetics of dPMP insertion opposite various templates have been determined. In one sequence context, the efficiency of dPMP insertion increases 4.6-fold opposite G < A << T < C, suggesting that the templating nucleotide modulates dPMP insertion efficiency by having to destack prior to dPTP binding. The efficiency of insertion of dPMP opposite T in the same sequence context increases 7-fold for primers terminating in G < A < C < T and is similar to that observed for nontemplated blunt-end extension, suggesting that stacking interactions between the pyrene and the primer terminus are also important. On heterogeneous templates, the average selectivity for dPMP insertion relative to the complementary dNMP decreases in the order of dAMP > dGMP > dTMP > dCMP, from a high of 5.8 when dAMP is to be inserted following a T to a low of 0.5 when dCMP is to be inserted following a C. The relative preference for dPMP insertion at a given site can be largely explained by the energetic cost of destacking the templating base and stacking of pyrene nucleotide relative to that of stacking and base pairing the complementary nucleotide. Thus, pyrene nucleotide represents a novel class of nucleotide-based chain-terminating DNA synthesis inhibitors whose base portion consists of a hydrophobic, non-hydrogen bonding, base-pair mimic.
Y家族DNA聚合酶酵母聚合酶η优先在无碱基位点、胸腺嘧啶二聚体的3'-T以及正常T的对面插入芘脱氧核糖单磷酸（dPMP），效率几乎相同。相比之下，诸如klenow片段和T7 DNA聚合酶等A家族聚合酶仅在无碱基位点和胸腺嘧啶二聚体的3'-T对面有效插入dPMP，而在未受损的DNA对面则不能有效插入。芘核苷酸也是聚合酶η介导的DNA合成的有效链终止抑制剂，但不是klenow片段或T7 DNA聚合酶的抑制剂。为了更好地理解聚合酶η插入dPMP的效率和序列特异性的起源，已经确定了在各种模板对面插入dPMP的动力学。在一种序列背景下，dPMP插入的效率在G < A << T < C对面增加了4.6倍，这表明模板核苷酸在dPTP结合之前必须解堆叠，从而调节dPMP插入效率。在相同序列背景下，对于以G < A < C < T结尾的引物，dPMP在T对面插入的效率增加了7倍，并且与非模板平端延伸所观察到的效率相似，这表明芘与引物末端之间的堆叠相互作用也很重要。在异质模板上，相对于互补dNMP，dPMP插入的平均选择性按dAMP > dGMP > dTMP > dCMP的顺序降低，从在T之后插入dAMP时的高达5.8到在C之后插入dCMP时的低至0.5。在给定位点对dPMP插入的相对偏好很大程度上可以通过模板碱基解堆叠以及芘核苷酸堆叠相对于互补核苷酸堆叠和碱基配对的能量成本来解释。因此，芘核苷酸代表了一类新型的基于核苷酸的链终止DNA合成抑制剂，其碱基部分由疏水、非氢键、碱基对模拟物组成。
