Side Chains That Influence Fidelity at the Polymerase Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Minnick, Dana T.; Bębenek, Katarzyna; Osheroff, Wendy P.; Turner, Robert; Astatke, Mekbib; Liu, Lixing; Kunkel, Thomas A.; Joyce, Catherine M.

doi:10.1074/jbc.274.5.3067

Cited by 129 publications

(162 citation statements)

References 34 publications

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“…The difference in k pol ratios may imply somewhat different transition states which are beyond our capability of modeling. Similar position dependent observations have been reported for the Klenow Fragment (35,36). Our results with the L561A variant are consistent with the accepted notion that active-site geometry is involved in base selection.…”

Section: The Kinetic Behavior Of the Rb69 Pol L561a Variantsupporting

confidence: 92%

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

et al. 2006

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Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to non-complementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k pol and K D values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k pol / K D ) for mispaired bases relative to the wild type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 × 10 −5 µM −1 s −1 for dCMP opposite templating C to 2 × 10 −3 µM −1 s −1 for dAMP opposite templating C. These k pol /K D values are 3-60 fold greater than those observed with the wild type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting E. coli, harboring a plasmid encoding the L561A variant of RB69 pol, with T4 phage bearing a mutant rII locus and the rates of reversions to rII + were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo − mutator activities. Keywords base selectivity; transient-state kinetic parameters; mutation frequency; modeling of mismatches DNA polymerase is the central component of replicases that are responsible for faithfully copying DNA. Despite the fact that all four dNTPs are potential substrates, replicative DNA polymerases are able to limit the incorporation of mismatched bases to about one in a million [for reviews see (1-8)]. Although this error frequency is acceptable for phage replication, it is still far too high to maintain genetic integrity during cell proliferation in † Supported by National Institutes of Health, Public Health Service grants GM63276 (to W.K.) and TW006626 (to A.B.). J.W.D. was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. * To whom correspondence and reprint requests should be addressed. telephone, (203) 785-4599; fax, (203) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript more complex organisms. To increase fidelity, cells have a number of mechanisms for correcting errors, including the ability of the replicative DNA polymerase itself, or an associated subunit, to excise misincorporated bases. In addition, there are specialized DNA polymerases that confront DNA damage inflicted by radiation, oxidation, alkylating agents, etc. which block the progress of replic...

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Section: The Kinetic Behavior Of the Rb69 Pol L561a Variantsupporting

confidence: 92%

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

et al. 2006

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“…Exo-free pol I was chosen for comparison wtih pol θ because both are A-family DNA polymerases (pol I is the founding member) and neither protein has exonuclease activities. The Klenow fragment of pol I is able to extend mismatches to some extent in some sequence contexts [16]. In the present experiments, exo-free pol I discriminated considerably better between paired and mispaired termini than did pol θ ( Fig.…”

Section: Pol θ Extends Mismatched Primer-terminisupporting

confidence: 50%

DNA polymerase θ (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

Seki

Wood

2008

DNA Repair

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DNA polymerase θ (pol θ) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol θ and of Polq-defective mice have suggested that pol θ participates in DNA damage tolerance. For example, pol θ was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol θ extended relatively efficiently from matched termini as well as termini with A:G, A:T, and A:C mismatches, with less descrimination than a well-studied A family DNA polymerase, exonuclease-free pol I from E. coli. Although pol θ was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6-4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol θ was combined with DNA polymerase ι , an enzyme that can insert a base opposite a UV-induced (6-4) photoproduct, complete bypass of a (6-4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol θ is proficient at extension of unpaired termini. These results show the potential of pol θ to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol θ in somatic mutagenesis and genome instability.

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“…Both biochemical and structural studies of family A DNA polymerases support the possibility that amino acid residues required for enzyme function lie outside the catalytic active site regions (19,(33)(34)(35)(36). Because structural information on pol ␥-␣ is very limited, a secondary structure prediction algorithm (PredictProtein) was used to identify the positions of potential ␣-helices and ␤-strands in the spacer region, and this information was taken into consideration in the generation of triple and nested single alanine mutations that were introduced as substitutions for highly conserved residues in the ␥1, ␥3, and ␥4 elements (Fig.…”

Section: Amino Acid Residues Important For Dna Polymerase Activitymentioning

confidence: 99%

mentioning

confidence: 99%

“…The pol domain comprises palm, fingers, and thumb subdomains that are separated from the 3Ј-5Ј exonuclease (exo) domain by a cleft that binds the template-primer. Whereas the template strand lies along the cleft between the two domains, the 3Ј-end of the primer (or nascent DNA strand) shuttles between them during proofreading DNA synthesis (18,19).Drosophila pol ␥ is a heterodimeric protein comprising catalytic and accessory subunits (20,21). The catalytic subunit contains the DNA polymerase and 3Ј-5Ј exonuclease activities (22), and the accessory subunit contributes to both the structural integrity and catalytic efficiency of the holoenzyme (21, 23).…”

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confidence: 99%

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Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

Luo

Kaguni

2005

Journal of Biological Chemistry

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The catalytic subunit (␣) of mitochondrial DNA polymerase (pol ␥) shares conserved DNA polymerase and 3 -5 exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol ␥-␣. Four ␥-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol ␥. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial singlestranded DNA-binding protein. Based on these results and crystallographic evidence showing that the templateprimer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol ␥ may position the substrate with respect to the enzyme catalytic domains.The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for energy production. Disruption of mitochondrial energy metabolism can occur by genetic or biochemical mechanisms and is associated with human disorders including degenerative diseases, cancer, and aging (1). Mutations in both the mitochondrial genome and in nuclear genes whose products have mitochondrial functions are linked to mitochondrial disease syndromes. Nuclear genes include those encoding the adenine nucleotide translocator 1 (2), thymidine phosphorylase (3), mitochondrial DNA helicase (Twinkle) (4), and the catalytic subunit of mitochondrial DNA polymerase (pol 1 ␥) (5). Pol ␥ has also been shown to be a target of oxidative damage, which reduces DNA polymerase activity in the mitochondrial matrix (6).Pol ␥ is the only DNA polymerase known to be required for mitochondrial DNA replication in animals (7). It is a member of the family A DNA polymerase group (8, 9), of which Escherichia coli pol I is the prototype (10). The crystal structures of numerous family A DNA polymerases have been solved, revealing a high degree of structural similarity among its members (11)(12)(13)(14)(15)(16)(17). The interaction between DNA polymerase and template-primer DNA has also been revealed in molecular detail by structural determination of pol:DNA co-crystals and in biochemical studies. The pol domain comprises palm, fingers, and thumb subdomains that are separated from the 3Ј-5Ј exonuclease (exo) domain by a cleft that binds the template-primer. Wher...

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Side Chains That Influence Fidelity at the Polymerase Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Cited by 129 publications

References 34 publications

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

DNA polymerase θ (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

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