Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans.
Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans.
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DOI：
10.1371/journal.pgen.1005049
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发表时间：
2015-03
期刊：
影响因子：
4.5
通讯作者：
Crouse GF
中科院分区：
文献类型：
--
作者：
Flood CL;Rodriguez GP;Bao G;Shockley AH;Kow YW;Crouse GF
现在已经确定，在酵母中，以及可能大多数真核生物中，最开始的dna复制是由dna聚合酶ε进行的，而滞后链的dna复制是由dna聚合酶δ进行的。然而，POLδ在前导链复制中的作用尚不确定。在这项工作中，我们使用酿酒酵母中的报告系统来测量特定碱基对的突变率，以确定POLε或POLδ杂合或纯合校对缺陷突变体在二倍体菌株中的影响。我们发现野生型的POLε分子不能校对缺陷的POLε分子产生的错误，而POLδ不仅可以校对缺陷的POLδ分子产生的错误，而且可以校正POLε缺陷分子产生的错误。这些结果表明，前导链上的脱氧核糖核酸合成的任何中断都可能导致POLδ完成，也解释了为什么POLδ校对突变体比POLε校对缺陷突变体有更高的突变率。对于通过AT→GC、TA→GC、CG→AT和GC→AT突变恢复的菌株，我们还发现基因方向对校对缺陷菌株的突变率有很强的影响，并证明这种方向依赖性很大程度上是由于错配延长的差异效率所致。我们还发现，与3‘端G不同，3’端的8oxoG与A相对有效地延伸，并且不受校对的影响。校对突变已被证明在小鼠和人类中都会导致肿瘤的形成；这里提出的结果可以帮助解释这些校对突变所表现出的特性。许多DNA聚合酶能够纠正它们的错误：在加入错误的碱基后，产生的错配会激活聚合酶的核酸外切酶活性，从而移除错配的碱基，使复制继续进行。因此，取消校对活动会导致更高的突变率。我们证明了酵母中两种主要的复制DNA聚合酶，POLδ和POLε，具有不同的校对能力。在二倍体细胞中，POLε不能纠正其他POLε分子造成的错误，而POLδ不仅能纠正其他POLδ分子造成的错误，还能纠正POLε分子造成的错误。我们还发现，未经校对纠正的错配碱基有很大不同的扩展可能性，这取决于特定的碱基错配。在人类中，POLδ或POLε校对缺陷会导致癌症，这些结果有助于解释这些肿瘤的形成，并发现POLε突变似乎与POLδ突变一样频繁或更频繁地出现在人类肿瘤中。
It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants. Many DNA polymerases are able to proofread their errors: after incorporation of a wrong base, the resulting mispair invokes an exonuclease activity of the polymerase that removes the mispaired base and allows replication to continue. Elimination of the proofreading activity thus results in much higher mutation rates. We demonstrate that the two major replicative DNA polymerases in yeast, Pol δ and Pol ε, have different proofreading abilities. In diploid cells, Pol ε is not able to proofread errors created by other Pol ε molecules, whereas Pol δ can proofread not only errors created by other Pol δ molecules but also errors created by Pol ε molecules. We also find that mispaired bases not corrected by proofreading have much different likelihoods of being extended, depending on the particular base-base mismatch. In humans, defects in Pol δ or Pol ε proofreading can lead to cancer, and these results help explain the formation of those tumors and the finding that Pol ε mutants seem to be found as frequently, or more so, in human tumors as Pol δ mutants.
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影响因子：
16
作者：
Ghodgaonkar, Medini Manohar;Lazzaro, Federico;Olivera-Pimentel, Maite;Artola-Boran, Mariela;Cejka, Petr;Reijns, Martin A.;Jackson, Andrew P.;Plevani, Paolo;Muzi-Falconi, Marco;Jiricny, Josef
通讯作者：
Jiricny, Josef
影响因子：
14.9
作者：
Carr PA;Wang HH;Sterling B;Isaacs FJ;Lajoie MJ;Xu G;Church GM;Jacobson JM
通讯作者：
Jacobson JM
影响因子：
4.8
作者：
Gabbai, Carolina B.;Yeeles, Joseph T. P.;Marians, Kenneth J.
通讯作者：
Marians, Kenneth J.
影响因子：
3.5
作者：
Church DN;Briggs SE;Palles C;Domingo E;Kearsey SJ;Grimes JM;Gorman M;Martin L;Howarth KM;Hodgson SV;NSECG Collaborators;Kaur K;Taylor J;Tomlinson IP
通讯作者：
Tomlinson IP
影响因子：
14.9
作者：
Aarts M;te Riele H
通讯作者：
te Riele H