Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

Carver, Theodore E.; Hochstrasser, R. M.; Millar, David P.

doi:10.1073/pnas.91.22.10670

Cited by 77 publications

(143 citation statements)

References 25 publications

Supporting

Mentioning

123

Contrasting

Order By: Relevance

“…Interestingly, Ϸ1% of the polymerase domain binding events were observed to directly transfer the primer from the polymerase domain to the exonuclease domain without prior dissociation from the DNA. Our results yield an intermediary level of exonuclease partitioning relative to previously published data (22)(23)(24) and are consistent with the idea that partitioning to the exonuclease site is increased in the presence of a terminally mismatched primer/ template.…”

Section: Resultssupporting

confidence: 80%

“…Analysis of the binding of KF to a fully paired primer/template showed FRET ratios suggesting Ϸ65% of primers were bound to the polymerase active site and Ϸ35% to the exonuclease site. These results are in agreement with prior studies that used fluorescence to measure the binding position of the primer in KF binary complexes (22)(23)(24). Measurements made during the incorporation of three dNTPs detected the movement of KF on the template in three discrete steps that correlated with the FRET ratios for the binding of the KF to primer/templates in which the primers had these three lengths.…”

Section: Figsupporting

confidence: 80%

“…To test whether the observed dynamics represent positioning the primer terminus in the exonuclease site, we used an analogous 16-mer/28-mer primer/template complex with a terminal G:A mismatch at the 3Ј-end, which is known to increase the binding of the primer/ template to the exonuclease domain (22). A characteristic smFRET trajectory for binding to this template shows the predominant presence of a state at Ϸ0.26 FRET (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Christian

Romano

Rueda

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3 -hydroxyl of the growing DNA chain. Here we report a singlemolecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Fö rster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.Klenow Fragment ͉ polymerase and exonuclease site ͉ single molecule fluorescence ͉ single nucleotide resolution ͉ structural dynamics T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1, 2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. The fingers subdomain is primarily involved in interactions with the singlestranded region of the DNA template and the incoming nucleotide; the palm forms the active site of the polymerase upon interaction with the incoming dNTP; and the thumb is responsible for binding double-stranded DNA. The exonuclease domain, located Ϸ30 Å from the polymerase domain, binds to the 3Ј-terminus of the primer when a mismatched base is incorporated (3).Like other high-fidelity polymerases, KF achieves its extraordinary accuracy through a series of steps that discriminate between a correct and incorrect dNTP. A minimal reaction pathway for KF has been proposed with much of the data obtained from chemical quench experiments (4, 5) (Fig. 1A). The rate-limiting step (k 3 ) that precedes the phosphoryl-transfer step (k 4 ) had been tentatively attributed to a conformational change of the fingers domain (6). A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps th...

show abstract

Section: Resultssupporting

confidence: 80%

Section: Figsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Christian

Romano

Rueda

2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Although the steady-state kinetics of nucleotide insertion opposite the lesion have been studied extensively for many DNA adducts (31)(32)(33)(34), data dealing with the long-range effects of a lesion on TLS are limited (35)(36)(37)(38)(39). Lindsley and Fuchs (36) have shown that the rate of primer extension by T7 DNA polymerase both at (n) and adjacent to (n+1) the lesion site with a DNA template containing an AF-lesion is reduced significantly (~10 −4 ) relative to unmodified control DNA.…”

Section: And N-(2′-deoxyguanosin-8-yl)-2-aminofluorene (Af)mentioning

confidence: 99%

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

Meneni¹,

Liang²,

Cho³

2007

Journal of Molecular Biology

View full text Add to dashboard Cite

SUMMARYAdduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2′-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the 'B-DNA' major groove, S is 'stacked' into the helix with base-displacement, and W is 'wedged' into the minor groove. In the present study, we conducted 19 F NMR, CD, Tm, and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B-or S-conformers while the FAF-G:A mismatch ones preferred the B-and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and Tm results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/ translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller and Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.

show abstract

“…Another difference between the mutant and wild type enzymes was that while A-T base pairs upstream of the 2AP mismatch, compared with upstream G-C base pairs, stimulated excision rates by the wild type T4 DNA polymerase, the A ϩ T or G ϩ C richness of the flanking DNA did not affect excision rates by the G255S-DNA polymerase. A ϩ T-rich sequences are expected to facilitate local melting (16,17); yet, the G255S-DNA polymerase, unlike the wild type enzyme, could not take advantage of A ϩ T richness to separate the primer strand from the template strand. These results are most consistent with a recent crystallographic study of the exonuclease domain showing the location of Gly-255 in a critical loop structure (14) and support a proofreading mechanism in which amino acid residues in the exonuclease domain of the T4 DNA polymerase assist the strand separation process.…”

mentioning

confidence: 99%

Using 2-Aminopurine Fluorescence and Mutational Analysis to Demonstrate an Active Role of Bacteriophage T4 DNA Polymerase in Strand Separation Required for 3′→ 5′-Exonuclease Activity

Marquez

Reha-Krantz

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3 3 5 exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G ؉ C-or A ؉ T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.Exonucleolytic proofreading by DNA polymerases is an important component of high fidelity DNA replication. Nucleotide insertion errors occur at a frequency of 10 Ϫ3 to 10 Ϫ5 , but the DNA polymerase-associated 3Ј 3 5Ј-exonuclease activity further reduces replication errors by 100-fold or more (reviewed in Ref. 1). Different steps of the proofreading reaction have been revealed by mutational analysis of the bacteriophage T4 DNA polymerase. Mutant T4 DNA polymerases deficient in 3Ј 3 5Ј-exonuclease activity exhibit a strong mutator phenotype that can be used to select for mutant DNA polymerases (2-4). The loss of the 3Ј 3 5Ј-exonuclease activity by mutation can be achieved by preventing distinct steps in the proofreading process. The hydrolysis reaction may be prevented if residues required for catalysis are substituted with other amino acids. For example, the substitution of Ala residues for Asp residues that bind essential Mg 2ϩ ions in the exonuclease active center reduces the exonuclease activity to barely detectable levels (5-7). Another reaction step in proofreading that may be affected by mutation is the translocation of the primer end of the DNA between the spatially distinct polymerase and exonuclease active centers. Identification of several "active-site switching mutants" has been described recently (8, 9). DNA polymerase proofreading is a dynamic process; if "switching" to the exonuclease active site is reduced by mutation, there is less opportunity for proofreading and a mutator phenotype is produced (9).Another proofreading reaction step that may be probed by mutational analysis of T4 DNA polymerase is strand separation. The 3Ј 3 5Ј-exonuclease activity o...

show abstract

Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

Cited by 77 publications

References 25 publications

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

Using 2-Aminopurine Fluorescence and Mutational Analysis to Demonstrate an Active Role of Bacteriophage T4 DNA Polymerase in Strand Separation Required for 3′→ 5′-Exonuclease Activity

Contact Info

Product

Resources

About