Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2-4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6-9-nucleotides from the primer terminus. We also subjected primers containing 3′ penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.