DNA Replication Fidelity

Kunkel, Thomas A.; Bębenek, Katarzyna

doi:10.1146/annurev.biochem.69.1.497

Cited by 1,051 publications

(985 citation statements)

References 219 publications

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“…This conclusion is consistent with crystallographic data showing a 0.6-1.8 Å displacement of the phosphodiester backbone at the site of a single tT insertion in a standard B-form DNA helix, 10 and with crystal structures of primer/ template/polymerase complexes. 11, 12 Results very similar to those reported here have also been obtained in the Herdewijn laboratory. 13 In summary, we have identified Deep Vent (exo-) as a polymerase that is capable of significant TNA synthesis.…”

supporting

confidence: 87%

TNA Synthesis by DNA Polymerases

2003

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The systematic evaluation of structures related to RNA 1 led to the recent discovery that (3′,2′)-R-L-threose nucleic acid (TNA) oligonucleotides are capable of Watson-Crick base-pairing with complementary DNA, RNA, and TNA oligonucleotides. 2 These observations, coupled with the chemical simplicity of threose relative to ribose, make TNA very interesting as a potential progenitor of RNA. 3 For this reason, we would like to compare the functional properties of TNA and RNA by attempting to evolve TNA aptamers and catalysts by in vitro selection. 4,5 This will require polymerases capable of copying a library of random DNA sequences into a library of TNA sequences for selection, and then copying the surviving TNA sequences back into DNA for amplification by PCR. We have recently identified several DNA polymerases capable of faithfully copying a short TNA template into DNA. 6 We now wish to report that some DNA polymerases are also capable of TNA synthesis on a DNA template when given the appropriate TNA triphosphate. These enzymes are candidates for directed evolution into more efficient DNA-dependent TNA polymerases.We used a primer extension assay to compare the ability of a set of DNA polymerases to synthesize TNA by extension of a DNA primer annealed to a DNA template, in the presence of chemically synthesized R-L-threofuranosyl thymidine-3′-triphosphate 1 (tTTP, Figure 1A). A synthetic DNA primer was annealed to either of two DNA templates in which the three template bases following the primer were adenine (A) or diaminopurine (D) (Figure 1b).We screened several different types of DNA polymerases including the Klenow fragment of DNA pol I (exo-), the thermophilic DNA polymerases Taq, Bst pol I, and Deep Vent (exo-), the bacteriophage T7 (exo-), and its commercial version Sequenase, the viral reverse transcriptases HIV RT, MMLV, and its mutated commercial version Superscript II, as well as the repair polymerases Pol , DinB, and Dbh (data not shown). All enzymes tested except DinB and Dbh were able to extend all of the DNA primer by at least one tT nucleotide. The DNA polymerases Bst pol I, Deep Vent (exo-), and HIV RT were capable of catalyzing multiple tT additions to the DNA primer ( Figure 2). Deep Vent (exo-) was by far the most effective at TNA synthesis (lanes 5-7). The HIV reverse transcriptase is unusual in catalyzing the rapid incorporation of two successive tT residues, but showing virtually no subsequent incorporation even with longer reaction times and additional polymerase. Only for Deep Vent (exo-) did the substitution of adenosine by diaminopurine in the template significantly enhance TNA synthesis, leading to quantitative incorporation of three tT residues. This substitution has previously been shown to increase the stability of DNA/TNA heteroduplexes and the efficiency of TNA-directed nonenzymatic ligation. 7 To understand the various factors that influence the rate of TNA synthesis, we have examined the catalytic efficiency of three aspects of the TNA synthesis reaction: (1) extension of a DN...

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confidence: 87%

TNA Synthesis by DNA Polymerases

2003

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“…In general, repair polymerases typically extend mismatches at a similar rate at which they incorporate mismatched nucleotides (14,15). One exception to this rule is Pol , which extends mismatches more frequently than it misincorporates them, and thus it has been proposed that the in vivo role of Pol is to extend from mismatches (30).…”

Section: S Pyogenes Dnae Extends Mismatches At a Similar Efficiency mentioning

confidence: 99%

“…Products were resolved on a 12% polyacrylamide (8 M urea) gel. Primer extension reactions were performed on primer templates having 5Ј homopolymer extensions of poly(T) (A, lanes 1-6), poly(dC) (B, lanes 7-12), or a poly(T) extension containing two dC residues at positions 6 and 12 (C, lanes [13][14][15][16][17][18]. The arrows on the left side of the gel (B) indicate the position of the 2 Cs on the template.…”

Section: S Pyogenes Dnae Efficiently Bypasses 8-oxo-dg and 2-aminopumentioning

confidence: 99%

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The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

Bruck

Goodman

O'Donnell

2003

Journal of Biological Chemistry

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DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (؊1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.

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“…Our approach in understanding the mechanism is to examine how this adduct may affect in vitro replication processes using mammalian polymerases, which are a key factor in maintaining genomic integrity. There are a number of human DNA-directed polymerases which have been identified as having lesion-bypassing capacity in addition to their various roles in cellular functions (for reviews see refs (11,12). Polymerase α (pol α) exhibits high fidelity, and is a moderately processive enzyme (13), functioning in both DNA replication and repair.…”

Section: Introductionmentioning

confidence: 99%

8-(Hydroxymethyl)-3,N⁴-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

Medina

Zhang

et al. 2002

Biochemistry

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DNA Replication Fidelity

Cited by 1,051 publications

References 219 publications

TNA Synthesis by DNA Polymerases

TNA Synthesis by DNA Polymerases

The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

8-(Hydroxymethyl)-3,N⁴-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

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DNA Replication Fidelity

Cited by 1,051 publications

References 219 publications

TNA Synthesis by DNA Polymerases

TNA Synthesis by DNA Polymerases

The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

8-(Hydroxymethyl)-3,N4-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

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8-(Hydroxymethyl)-3,N⁴-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases