Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1-5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3' end are partially corrected and extended. Indels located 7-9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.

中文翻译：

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通过MALDI-TOF质谱分析法分析的单核苷酸插入/缺失复制错误的校对。

小核苷酸插入/缺失（插入/缺失）错误是DNA合成中常见的复制错误之一。据认为，最常见的插入缺失错误是由于重复序列在DNA复制过程中易于滑动。校对和DNA错配修复是indel错误校正中的重要因素，以保持遗传信息交易的高保真度。我们采用MALDI-TOF质谱（MS）分析来测量Klenow聚合酶（KF）校正插入缺失错误的效率。在此，将未标记的和非放射性同位素的寡核苷酸引物退火至形成单核苷酸插入缺失的模板DNA，并在不同脱氧核糖核苷酸三磷酸和/或双脱氧核糖核苷酸三磷酸的组合存在下通过KF校对。通过KF修饰的引物质量变化来鉴定校对产品。我们检查了在引物-模板连接的各个位置上包含插入缺失错误的DNA的校对。我们发现位于引物末端1-5个核苷酸（nt）的插入缺失可被有效地校对，而距3'末端6-nt的插入/缺失可得到部分校正和扩展。位于引物末端7-9-nt的插入缺失逃避了校对，并被聚合酶延长。通过结构分析，在聚合酶和引物-模板连接相互作用的背景下讨论了这些观察结果的可能的潜在机制。我们发现位于引物末端1-5个核苷酸（nt）的插入缺失可被有效地校对，而距3'末端6-nt的插入/缺失可得到部分校正和扩展。位于引物末端7-9-nt的插入缺失逃避了校对，并被聚合酶延长。通过结构分析，在聚合酶和引物-模板连接相互作用的背景下讨论了这些观察结果的可能的潜在机制。我们发现位于引物末端1-5个核苷酸（nt）的插入缺失可被有效地校对，而距3'末端6-nt的插入/缺失可得到部分校正和扩展。位于引物末端7-9-nt的插入缺失逃避了校对，并被聚合酶延长。通过结构分析，在聚合酶和引物-模板连接相互作用的背景下讨论了这些观察结果的可能的潜在机制。