• Identification of a premature termination of DNA polymerization in vitro by Klenow fragment mutants

    • Fulltext


        Click here to view fulltext PDF

      Permanent link:

    • Keywords


      Blunt end; DNA polymerase; Klenow fragment; sticky end

    • Abstract


      DNA polymerization products by Klenow fragment (KF) are blunt-ended. In the present study, we found that the Klenow fragment mutants with partial deletions of thumb subdomain were unable to extend primers to the 5′ terminal of templates, thus creating 5′ overhanging sticky ends 2 nt long. We termed this phenomenon as PmTP (premature termination of polymerization). The KF mutants produced homogenous sticky-ended products only under mild reaction conditions, whereas under vigorous reaction conditions, the sticky ends were prone to be blunt-ended. It was also identified that deletions of more than four residues of KF thumb subdomain could induce PmTP, and two-residue deletion of KF thumb subdomain only induced PmTP in a lower-concentration situation. Structure modelling analysis suggested that shortening or destruction of 𝛼 helix H1 at the tip of the thumb subdomain was crucial to PmTP, while the conserved residues in front of 𝛼 helix was less important. PmTP might be caused by the reduced DNA-binding affinity of the mutants. The sticky ends made by PmTP have potential applications in gene splicing and molecular cloning techniques.

    • Author Affiliations


      Guojie Zhao1 Hua Wei2 Yifu Guan1

      1. Department of Biochemistry and Molecular Biology, Key Laboratory of Medical Cell Biology of Ministry of Education, China Medical University, Shenyang, China
      2. Animal Science and Veterinary Medicine College, Shenyang Agricultural University, Shenyang, China
    • Dates

    • Supplementary Material

  • Journal of Biosciences | News

      Forthcoming Special issue.

    • To trigger further research on plant mitochondria, the Journal of Biosciences is bringing out a special issue titled "Plant Mitochondria: Properties and Interactions with Other Organelles".

      Plant mitochondria are quite distinct and have unique features, such as a cyanide-insensitive alternate pathway. They also interact with chloroplasts to optimize photosynthetic carbon assimilation.

      Submissions are welcome until 30 July 2023. The contributions can be original articles, short communications, reviews, or mini-reviews on any topic related to plant mitochondria.

      Authors can submit their articles online at https://www.editorialmanager.com/jbsc/default2.aspx

      Posted on April 12, 2023
    • Editorial Note on Continuous Article Publication

      Posted on July 25, 2019

      Click here for Editorial Note on CAP Mode

© 2022-2023 Indian Academy of Sciences, Bengaluru.