Replication of non-hydrogen bonded bases by DNA polymerases: A mechanism for steric matching

Kool, Eric T.

doi:10.1002/(sici)1097-0282(1998)48:1<3::aid-bip2>3.0.co;2-7

Cited by 115 publications

(112 citation statements)

References 69 publications

Supporting

Mentioning

103

Contrasting

Unclassified

Order By: Relevance

“…These populations are consistent with the observed X-ray structure. In addition to rapid ring flipping, additional self pair motion is suggested by line broadening of intranucleotide NOE cross peaks of the flanking nucleobases dA 5 and dG 10 , and dA 5 imino resonance broadening which may result from enhanced solvent exchange, possibly due to increased solvent accessibility or base pair fluctuations. 38 Such fluctuations may be coupled to the observed ring flipping of the d3FB analogs.…”

Section: Resultsmentioning

confidence: 99%

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

et al. 2007

View full text Add to dashboard Cite

Expansion of the genetic alphabet has been a long time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semi-synthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogs, such as propinyl isocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogs inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogs that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluoro benzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self pair formed between two d3FB analogs is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analog (dBEN). In all, the data suggest that structure, electrostatics and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.

show abstract

Section: Resultsmentioning

confidence: 99%

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

et al. 2007

View full text Add to dashboard Cite

show abstract

“…This would rationalize the high error rates for three mismatches involving misinsertion of dATP, the most hydrophobic base. It might even explain the unusually high error rates for pyrimidine-pyrimidine mismatches, which have been suggested to be more readily accommodated if both pyrimidines can be desolvated, thus allowing these smallest of the 12 possible base-base mismatches to more easily escape geometric selection in the nascent base pair binding pocket (65). Region II of Pol ⑀ also differs from that in other family B members in containing a 66-amino acid insertion that is predicted to encode two ␣-helices (Fig.…”

Section: Discussionmentioning

confidence: 99%

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Shcherbakova

Pavlov

Chilkova

et al. 2003

Journal of Biological Chemistry

115

137

View full text Add to dashboard Cite

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ⑀ (Pol ⑀) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ⑀ synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of <2 ؋ 10 ؊5 and <5 ؋ 10 ؊7 , respectively. Pol ⑀ lacking 3 3 5 exonuclease activity is less accurate to a degree suggesting that wild type Pol ⑀ proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ⑀ is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T⅐dTTP, T⅐dCTP, and C⅐dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ⑀ differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ⑀ in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ⑀ proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ⑀ polymerase activity in DNA replication.Replication of chromosomes in eukaryotes is believed to require three DNA polymerases, Pol ␣, 1 Pol ␦, and Pol ⑀ (for reviews, see Refs. 1-3). Pol ␣ has an associated DNA primase activity and synthesizes short RNA-DNA primers to initiate replication at origins and start Okazaki fragments on the lagging DNA strand. The roles of Pol ␦ and Pol ⑀ in chromosomal replication are not clearly defined. Evidence for essential roles of Pol ␦ and Pol ⑀ in replication comes from genetic studies in Saccharomyces cerevisiae. Mutational inactivation of catalytic subunits and some of the additional subunits of both polymerases is lethal. Strains carrying temperature-sensitive mutations in POL3 gene (encoding the catalytic subunit of Pol ␦), POL2, or DPB2 genes (encoding subunits of Pol ⑀) arrest in S phase of the cell cycle with a terminal morphology characteristic of a DNA replication defect upon shift to non-permissive temperature (4 -6). Incorporation of labeled precursors into DNA stops in these mutants at non-permissive temperature (4, 5, 7). The POL3, POL2, and DPB2 genes, as well as the DPB3 gene encoding the third subunit of Pol ⑀, are expressed periodically in the cell cycle with a peak in G 1 to S phase transition (4, 5, 8), which is characteristic of genes encoding DNA replication proteins. Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9, 10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).To accommodate roles for Pol ␦ and Pol ⑀ at a replication fork, it was proposed...

show abstract

“…6 Since hydrogen bonds are apparently dispensable in maintaining natural enzyme activity, we proposed that the fidelity of DNA replication might arise in large part from steric matching of bases within a tightly confined active site. 3,7,8 This hypothesis was criticized by suggesting that the reason that F behaved as such an effective mimic of T was that it actually did form robust hydrogen bonds with adenine.9 After this, several experiments and calculations with this fluorinated analogue and related compounds have concluded that C-F…H-N hydrogen bonding character is quite poor in this context, especially in the aqueous environment.10 -18 Nonetheless, a clearer way to avoid the issue of possible hydrogen bonds between F and A is to replace adenine with a nonpolar isostere, as was done for thymine. To this end, we chose a 4-methylbenzimidazole replacement used for adenine (Z) and studied its ability to be replicated opposite F. 19 , 20 It was found, interestingly, that this pair was an efficient substrate for the Klenow polymerase, and it was processed with significant levels of selectivity.…”

Section: Introductionmentioning

confidence: 99%

“…A steric matching hypothesis for DNA replication 7 requires that to be a good substrate, a base pair (whether it consists of natural or nonnatural bases) must be able to adopt a geometry close to that of the standard pairs without steric clashes. In previous structural studies we found that an isolated difluorotoluene deoxynucleoside did not significantly distort a DNA duplex, even though when paired with adenine it significantly lowered the stability of the helix.…”

Section: Introductionmentioning

confidence: 99%

Solution Structure of a Nonpolar, Non-Hydrogen-Bonded Base Pair Surrogate in DNA

2000

Self Cite

View full text Add to dashboard Cite

We describe the structure in aqueous solution of a DNA duplex containing a base pair that is structurally analogous to A-T but which lacks hydrogen bonds. Base analogues F (a nonpolar isostere of thymine) and Z (a nonpolar isostere of adenine) are paired opposite one another in a 12 base pair duplex. The sequence context is the binding site of recently studied transcription factor hSRY. The Z-F pair has been shown to be replicated surprisingly well and selectively by DNA polymerase enzymes, considering that it is destabilizing and lacks Watson-Crick hydrogen bonds. The enzymatic studies led to the suggestion that part of the functional activity arises because the pair resembles a natural one in geometry. The present results show that, despite the absence of Watson-Crick hydrogen bonds, the Z-F pair structurally resembles an A-T pair in the same context. This lends support to the proposal that shape matching is an important component in replication, and suggests the general utility of using Z-F as a nonpolar replacement for A-T in probing protein-DNA interactions.

show abstract

Replication of non-hydrogen bonded bases by DNA polymerases: A mechanism for steric matching

Cited by 115 publications

References 69 publications

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Solution Structure of a Nonpolar, Non-Hydrogen-Bonded Base Pair Surrogate in DNA

Contact Info

Product

Resources

About