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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- DTT:
-
dithiothreitol
- IPTG:
-
isopropyl β-d-1-thiogalactopyranoside
- KF:
-
Klenow fragment
- KPB:
-
potassium phosphate buffer
- ODN:
-
oligodeoxyribonucleotide
- PAGE:
-
polyacrylamide gel electrophoresis
- PmTP:
-
premature termination of polymerization
- TdT:
-
terminal deoxynucleotidyl transferase
References
Arnold K, Bordoli L, Kopp J and Schwede T 2006 The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22 195–201
Bebenek K, Beard WA, Casas-Finet JR, Kim HR, Darden TA, Wilson SH and Kunkel TA 1995 Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain. J. Biol. Chem. 270 19516–19523
Bedford E, Tabor S and Richardson CC 1997 The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. Proc. Natl. Acad. Sci. USA 94 479–484
Beese LS, Derbyshire V and Steitz TA 1993 Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260 352–355
Carroll WL 1993 Introduction to recombinant-DNA technology. Am. J. Clin. Nutr. 58 249S–258S
Clark JM 1988 Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16 9677–9686
Costa GL and Weiner MP 1994 Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments. Nucleic Acids Res. 22 2423
Delagoutte E 2003 Function and Assembly of the Bacteriophage T4 DNA Replication Complex: Interactions of the T4 polymerase with various model DNA constructs. J. Biol. Chem. 278 25435–25447
Filee J, Forterre P, Sen-Lin T and Laurent J 2002 Evolution of DNA polymerase families: evidences for multiple gene exchange between cellular and viral proteins. J. Mol. Evol. 54 763–773
Horton RM, Cai ZL, Ho SN and Pease LR 1990 Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8 528–535
Hsu GW, Kiefer JR, Burnouf D, Becherel OJ, Fuchs RP and Beese LS 2004 Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase. J. Biol. Chem. 279 50280–50285
Kohlstaedt LA, Wang J, Friedman JM, Rice PA and Steitz TA 1992 Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256 1783–1790
Kroutil LC, Register K, Bebenek K and Kunkel TA 1996 Exonucleolytic proofreading during replication of repetitive DNA. Biochemistry 35 1046–1053
Li Y, Korolev S and Waksman G 1998 Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 17 7514–7525
Li Y, Dutta S, Doublie S, Bdour HM, Taylor JS and Ellenberger T 2004 Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7. Nat. Struct. Mol. Biol. 11 784–790
Minnick DT, Astatke M, Joyce CM and Kunkel TA 1996 A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity. J. Biol. Chem. 271 24954–24961
Minnick DT, Bebenek K, Osheroff WP, Turner RM Jr, Astatke M, Liu L, Kunkel TA and Joyce CM 1999 Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). J. Biol. Chem. 274 3067–3075
Motea EA and Berdis AJ 2010 Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase. Biochim. Biophys. Acta – Proteins Proteomics 1804 1151–1166
Ollis DL, Brick P, Hamlin R, Xuong NG and Steitz TA 1985 Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 313 762–766
Raymond FL, Whittaker J, Jenkins L, Lench N and Chitty LS 2010 Molecular prenatal diagnosis: the impact of modern technologies. Prenat. Diagn. 30 674–681
Schamhart DH and Westerhof AC 1999 Strategies for gene cloning. Urol Res 27 83–96
Turner RM Jr, Grindley ND and Joyce CM 2003 Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis. Biochemistry 42 2373–2385
Yang S, Li X, Ding D, Hou J, Jin Z, Yu X, Bo T, Li W and Li M 2005 A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase. Biotechnol. Appl. Biochem. 42 223–226
Yang XM and Richardson CC 1997 Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization. J. Biol. Chem. 272 6599–6606
Zhao G and Guan Y 2010 Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions. Acta Biochim. Biophys. Sin. 42 722–728
Zhou MY and Gomez-Sanchez CE 2000 Universal TA cloning. Curr. Iss. Mol. Biol. 2 1–7
Acknowledgements
This work was supported by a grant from the National Natural Science Foundation of China (No. 31070705).
Author information
Authors and Affiliations
Corresponding author
Additional information
Corresponding editor: B JAGADEESHWAR RAO
MS received 22 October 2012; accepted 22 February 2013
Corresponding editor: B Jagadeeshwar Rao
[Zhao G, Wei H and Guan Y 2013 Identification of a premature termination of DNA polymerization in vitro by Klenow fragment mutants. J. Biosci. 38 1–11] DOI 10.1007/s12038-013-9314-y
Supplementary materials pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/jun2013/supp/zhao.pdf
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 72.7 kb)
Rights and permissions
About this article
Cite this article
Zhao, G., Wei, H. & Guan, Y. Identification of a premature termination of DNA polymerization in vitro by Klenow fragment mutants. J Biosci 38, 279–289 (2013). https://doi.org/10.1007/s12038-013-9314-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12038-013-9314-y