The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site.
The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site.
复制标题
5-羟基胞嘧啶的错误编码可能性是由于复制聚合酶活性位点的模板不稳定而产生的。
DOI：
10.1021/bi201219s
复制
发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Doublié,Sylvie
中科院分区：
文献类型：
--
作者：
Zahn,KarlE;Averill,April;Wallace,SusanS;Doublié,Sylvie
5-羟基胞嘧啶 (5-OHC) 是一种稳定的胞嘧啶氧化产物，与 C→T 过渡突变频率增加相关。当这种损伤逃脱了碱基切除修复途径的识别并在 DNA 合成过程中持续充当模板碱基时，复制性 DNA 聚合酶通常会在引物末端错误地掺入 dAMP，这可能导致突变固定和随后的疾病。为了表征与 5-OHC 相对的 DNA 合成动力学，我们开始将未修饰的 dCMP 与 5-OHC、5-氟胞嘧啶 (5-FC) 和 5-甲基胞嘧啶 (5-MEC) 进行比较，其中这些碱基充当 RB69 gp43 活性位点的模板，RB69 gp43 是一种与人类复制 DNA 聚合酶具有同源性的高保真 DNA 聚合酶。这项研究展示了结合这种生理上重要的诱变前 DNA 损伤的任何 DNA 聚合酶的第一个晶体结构，表明虽然 dGMP 通过正常的 Watson-Crick 碱基配对被 5-OHC 稳定，但 dAMP 的掺入会导致模板解堆积和不稳定。此外，C5 取代基的电负性似乎对这些胞嘧啶样模板的错误编码潜力很重要。虽然 dAMP 相对 5-OHC 的掺入效率比相对的未修饰 dCMP 的效率高约 5 倍，但也观察到与 5-FC 相对的掺入水平升高，但与 5-MEC 不同。总而言之，这些数据表明 5-OHC 的不均匀模板是由于堆叠能力减弱所致，这使得 dAMP 掺入以类似于观察到的相反无碱基位点的方式进行。
5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C → T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson–Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ∼5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.