Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ε.
Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ε.
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DOI：
10.1016/j.dnarep.2011.02.001
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发表时间：
2011-05-05
期刊：
影响因子：
3.8
通讯作者：
Kunkel TA
中科院分区：
文献类型：
--
作者：
Clark AB;Lujan SA;Kissling GE;Kunkel TA
在利用体内存在的dNTP和rNTP浓度进行体外DNA合成的过程中，酵母复制DNA聚合酶α，δ和ε(POLS，α，δ和ε)稳定地将rNTPs整合到DNA中。RNTP也在体内复制过程中被掺入，并以RNaseH2依赖的方式修复。在编码POLε(Pol2-M644G)突变等位基因的菌株中，由于编码RNaseH2催化亚基的RNH201基因缺失，导致短重复序列中2-5个碱基对的缺失。缺失率取决于报告基因相对于附近复制起点的方向，这表明突变是由复制过程中加入的rNMP造成的。在这里，我们证明了在编码野生型Δ聚合酶的rnh201菌株中，2-5个碱基对的缺失突变也显著增加。与pol2-M644G菌株一样，缺失发生在重复序列上，并与方向有关，这表明涉及错配的链的错配可能会发生错配修复。然而，出乎意料的是，在pol2-M644G株中，由于RNH201缺失而导致的2-5个碱基对缺失率不受MSH3和/或MSH6缺失的影响。可能是错配修复机制不能修复由M644G POLε引入的未修复的rNMP引起的错配，但这种可能性被观察到Msh2-MSH6可以与含有核糖核苷酸的错配结合而被证明是错误的。或者，在复制期间由M644G POLε将rNMP掺入之后，未修复的rNMP向突变的转化可能发生在复制环境之外，例如，在修复由DNA中的rNMPs产生的缺口期间。研究结果做出了一些有趣的预测，这些预测可以得到检验。
During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ε (Pols α, δ and ε) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ε (pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2-5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2-5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2-5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ε, but this possibility is belied by the observation that Msh2-Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ε during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.
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DOI：
10.1073/pnas.94.5.1619
发表时间：
1997-03-04
影响因子：
11.1
作者：
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通讯作者：
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