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

      Guojie Zhao Hua Wei Yifu Guan

      Articles Volume 38 Issue 2 June 2013 pp 279-289

    • Fulltext

       

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      Permanent link:
      https://www.ias.ac.in/article/fulltext/jbsc/038/02/0279-0289

    • 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

       
      Published
      June 2013
      Manuscript received
      22 October 2012
      Accepted
      22 February 2013

    • Supplementary Material

       

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