Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases
Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases
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DOI：
10.1073/pnas.0914857107
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发表时间：
2010-03-16
影响因子：
11.1
通讯作者：
Kunkel, Thomas A.
中科院分区：
文献类型：
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作者：
McElhinny, Stephanie A. Nick;Watts, Brian E.;Kunkel, Thomas A.
对酿酒酵母中三磷酸核苷水平的测量表明，四种 rNTP 的摩尔过量是其相应 dNTP 的 36 至 190 倍。在使用生理三磷酸核苷浓度的体外 DNA 合成过程中，参与前导链复制的酵母 DNA 聚合酶 epsilon 每 1,250 个 dNMP 会掺入一个 rNMP。进行滞后链复制的 Pol δ 和 Pol α 分别每 5,000 或 625 dNMP 掺入 1 个 rNMP。对 rNMP 掺入的歧视差异很大，在某些情况下超过 100 倍，具体取决于碱基的身份及其所在的模板序列上下文。考虑到对 Pols α、δ 和 epsilon 催化的复制量的估计，结果与酵母每轮复制期间可能有超过 10,000 个 rNMP 掺入核基因组的可能性一致。因此，rNMP 可能是引入真核基因组的最常见的非规范核苷酸。讨论了丰富的核糖核苷酸掺入 DNA 的潜在有益和消极后果，包括 DNA 中未修复的 rNMP 可能会出现问题，因为酵母 DNA 聚合酶 epsilon 很难绕过 DNA 模板中存在的单个 rNMP。
Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.