Nucleic Acids Research Advance Access originally published online on October 28, 2006
Nucleic Acids Research 2006 34(20):6051-6063; doi:10.1093/nar/gkl769
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Nucleic Acids Research, 2006, Vol. 34, No. 20 6051-6063
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity
Instituto de Biología Molecular ‘Eladio Viñuela’ (CSIC), Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
*To whom correspondence should be addressed. Tel: +344 91 4978436; Fax: +34 91 4978490; Email: msalas@cbm.uam.es
Received July 21, 2006. Revised September 11, 2006. Accepted September 28, 2006.
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ABSTRACT |
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This paper shows that the protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1, unlike other DNA polymerases, do not require unwinding or processivity factors for efficient synthesis of full-length terminal protein (TP)-DNA. Analysis of their polymerization activity shows that both DNA polymerases base their replication efficiency on a high processivity and on the capacity to couple polymerization to strand displacement. Both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Additionally, Nf double-stranded DNA binding protein (DBP) greatly stimulated the in vitro formation of the TP-dAMP initiation complex by decreasing the Km value for dATP of the Nf DNA polymerase by >20-fold. Whereas Nf DNA polymerase, as the
29 enzyme, is able to use its homologous TP as well as DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. Such exceptional behaviour is discussed in the light of the recently solved structure of the DNA polymerase/TP complex of the related bacteriophage 29.
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INTRODUCTION |
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The inability of DNA polymerases to start de novo DNA synthesis imposes in most organisms the necessity of an RNA molecule to provide the 3'-OH group needed to initiate DNA elongation. This requirement creates a dilemma for the replication of the ends of linear genomes since, once the last RNA primer for the lagging strand synthesis is removed, a portion of ssDNA at the end of the genome will remain uncopied. In order to avoid the continuous shortening of the linear genomes in subsequent replication rounds, several mechanisms have evolved, most of them making use of the presence of repetitive sequences at the ends of the chromosomes that allow to create long concatemers, to circularize, or to form hairpin loops to fill the incomplete 5' ends. In higher eukaryotes, telomerase prevents chromosome ends shortening by elongating the 3'-OH group of the ssDNA end using as template its own RNA (
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MATERIALS AND METHODS |
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Nucleotides and DNAs
Proteins
3'–5' exonuclease assays
3'–5' Exonuclease activity on ssDNA
Hydrolysis of pNP-TMP
3'–5' Exonuclease assay on matched and mismatched primer-terminus
DNA gel retardation assay
Polymerase/3'–5' exonuclease (pol/exo) coupled assay
TP-primed initiation assay
Processivity and strand displacement assays
Replication of primed M13 DNA
Replication assay (protein-primed initiation plus elongation) with TP-DNA as template
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RESULTS |
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Nf and GA-1 DNA polymerases are provided with a 3'–5' exonuclease activity
3'–5' Exonuclease and polymerization activities of Nf and GA-1 DNA polymerases are coordinated
Nf DNA polymerase displays the capacity to couple strand displacement to polymerization
Protein-primed TP-DNA replication performed by Nf and GA-1 DNA polymerases
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DISCUSSION |
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DNA polymerase from bacteriophages Nf and GA-1
Nf and GA-1 DNA polymerases are proofreading enzymes
Nf and GA-1 DNA polymerases couple polymerization to strand displacement processively
GA-1 DNA polymerase, a paradigmatic enzyme
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