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

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 tworesidue 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 DNAbinding affinity of the mutants. The sticky ends made by PmTP have potential applications in gene splicing and molecular cloning techniques.