A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity - PubMed (original) (raw)
A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity
S Moran et al. Proc Natl Acad Sci U S A. 1997.
Abstract
Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.
Figures
Figure 1
The nucleotide structures and DNA sequences in this study. (A) Chemical structures of nucleosides T and analog F alongside space-filling models of the “nucleobases” of the two. (B) Sequences of template–primer duplexes used in steady-state studies. The standing-start substrate utilizes a 23-nt primer, whereas the running-start substrate utilizes an 18-nt primer.
Figure 2
Autoradiogram showing a survey of standing-start single-nucleotide insertions by the KF (exo−) enzyme, including all possible cases of analog F in the template and as a dNTP. The data were taken using 2 μM dNTP, and the reactions were stopped after 2 min.
Figure 3
Autoradiogram showing running-start primer elongations up to and beyond adenine with comparison with the known pause opposite an abasic nucleotide (φ). Reactions were performed with 20 μM of each dNTP; for the template containing A, the dNTP mixture was dATP + dCTP + dGTP + dFTP, and for the abasic template, dATP + dCTP + dGTP + dTTP.
Figure 4
Histogram showing accuracy of template selection for nucleotide insertion by the KF enzyme. Semilog plot for insertion efficiency of dFTP and dTTP opposite each of the four natural bases in a template.
Similar articles
- A specific partner for abasic damage in DNA.
Matray TJ, Kool ET. Matray TJ, et al. Nature. 1999 Jun 17;399(6737):704-8. doi: 10.1038/21453. Nature. 1999. PMID: 10385125 - The steric hypothesis for DNA replication and fluorine hydrogen bonding revisited in light of structural data.
Egli M. Egli M. Acc Chem Res. 2012 Aug 21;45(8):1237-46. doi: 10.1021/ar200303k. Epub 2012 Apr 23. Acc Chem Res. 2012. PMID: 22524491 Free PMC article. Review. - DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics.
Potapova O, Chan C, DeLucia AM, Helquist SA, Kool ET, Grindley ND, Joyce CM. Potapova O, et al. Biochemistry. 2006 Jan 24;45(3):890-8. doi: 10.1021/bi051792i. Biochemistry. 2006. PMID: 16411765 Free PMC article. - Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used.
Singer B, Chavez F, Goodman MF, Essigmann JM, Dosanjh MK. Singer B, et al. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8271-4. doi: 10.1073/pnas.86.21.8271. Proc Natl Acad Sci U S A. 1989. PMID: 2682644 Free PMC article.
Cited by
- Improved synthesis of the unnatural base NaM, and evaluation of its orthogonality in in vitro transcription and translation.
Le AV, Hartman MCT. Le AV, et al. RSC Chem Biol. 2024 Sep 11. doi: 10.1039/d4cb00121d. Online ahead of print. RSC Chem Biol. 2024. PMID: 39279876 Free PMC article. - Enzyme-assisted high throughput sequencing of an expanded genetic alphabet at single base resolution.
Wang B, Bradley KM, Kim MJ, Laos R, Chen C, Gerloff DL, Manfio L, Yang Z, Benner SA. Wang B, et al. Nat Commun. 2024 May 14;15(1):4057. doi: 10.1038/s41467-024-48408-9. Nat Commun. 2024. PMID: 38744910 Free PMC article. - Enzyme-Assisted High Throughput Sequencing of an Expanded Genetic Alphabet at Single Base Resolution.
Wang B, Bradley KM, Kim MJ, Laos R, Chen C, Gerloff DL, Manfio L, Yang Z, Benner SA. Wang B, et al. Res Sq [Preprint]. 2023 Dec 21:rs.3.rs-3678081. doi: 10.21203/rs.3.rs-3678081/v1. Res Sq. 2023. PMID: 38196584 Free PMC article. Updated. Preprint. - Nickel-Catalyzed Radical Mechanisms: Informing Cross-Coupling for Synthesizing Non-Canonical Biomolecules.
Dawson GA, Spielvogel EH, Diao T. Dawson GA, et al. Acc Chem Res. 2023 Dec 19;56(24):3640-3653. doi: 10.1021/acs.accounts.3c00588. Epub 2023 Nov 30. Acc Chem Res. 2023. PMID: 38033206 Free PMC article. - Complementary base pair interactions between different rare tautomers of the second-generation artificial genetic alphabets.
Jena NR, Das P, Shukla PK. Jena NR, et al. J Mol Model. 2023 Apr 4;29(5):125. doi: 10.1007/s00894-023-05537-0. J Mol Model. 2023. PMID: 37014428
References
- Kornberg A, Baker T A, editors. DNA Replication. 2nd Ed. New York: Freeman; 1992. p. 113.
- Loeb L A, Kunkel T A. Annu Rev Biochem. 1982;52:429. - PubMed
- Echols H, Goodman M F. Annu Rev Biochem. 1991;60:477–511. - PubMed
- Travaglini E C, Mildvan A S, Loeb L A. J Biol Chem. 1975;250:8647–8656. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous