Proofreading of single nucleotide insertion/deletion replication errors analyzed by MALDI-TOF mass spectrometry assay.

• A high resolution MALDI-TOF MS based assay were applied for the polI Klenow Fragment proofreading of indel errors. • Two approaches were used with reactions containing either 4 ddNTPs or 3 dNTPs to analyze proofreading products in detail. • KF proofreads Indel errors located 1–5-nucleotides (nt) from the primer terminus with high efficiency. • Indel errors located 7-9-nt from the primer terminus escape proofreading and are elongated by KF. 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. [ABSTRACT FROM AUTHOR]