Proofreading of mismatches within primer-template junctions by Escherichia coli DNA polymerase I in vitro and in vivo

Hui-Lan Chang; Kang-Yi Su; Steven D Goodman; Yung-Chu Chuang; Shen-Jyue Hsu; Yi-Kai Fang; Hsiao-Pei Yu; Cheng-Hao Fang; Ya-Chien Yang; Sui-Yuan Chang; Woei-Horng Fang

doi:10.1016/j.dnarep.2025.103864

Proofreading of mismatches within primer-template junctions by Escherichia coli DNA polymerase I in vitro and in vivo

DNA Repair (Amst). 2025 Jun 23:152:103864. doi: 10.1016/j.dnarep.2025.103864. Online ahead of print.

Authors

Affiliations

¹ Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 100229, Taiwan, ROC.
² Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 100229, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 10063, Taiwan, ROC.
³ Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, the Ohio State University, Columbus, OH, USA.
⁴ Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 100229, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 10063, Taiwan, ROC. Electronic address: Sychang@ntu.edu.tw.
⁵ Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 100229, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 10063, Taiwan, ROC. Electronic address: whfang@ntu.edu.tw.

PMID: 40577992
DOI: 10.1016/j.dnarep.2025.103864

Abstract

Escherichia coli DNA polymerase I (Pol I) possesses a 3' to 5' proofreading function. Using a non-inhibitory in vitro proofreading assay and MALDI-TOF MS analysis, we demonstrated the Pol I proofreading function was effective at removal of mismatches within the primer-template junction. Mismatches of 1-4 nucleotides (nt) from the primer 3' end could be completely or partially corrected, with no additional editing observed further upstream. A backward movement mechanism was proposed involving distributive backtracking of polymerase along the template to remove non-fully complemented primers in order for DNA synthesis to recover. Co-editing DNA substrates containing two mismatches, one at 1-4-nt of the primer 3' end and the other outside of normal proofreading range, confirmed our distributive backtracking hypothesis. Additionally, a time course analysis revealed proofreading of internal mismatches was a non-processive reaction. To further confirm the validity of our proofreading model, we used in vivo, phagemid-derived nicked C-C substrates. Transformation results were consistent with the notion that mismatches located less than 4-nt upstream of the 3' end could be successfully proofread. In vivo proofreading of double mismatches also supports our model of polymerase backtracking for internal mismatch editing.

Keywords: DNA mismatch; DNA polymerase I; DNA replication error; In vivo transformation assay; MALDI MS analysis; Proofreading.