Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing

Liu, Dongyu; Moran, Sean D.; Kool, Eric T.

doi:10.1016/s1074-5521(97)90300-8

Cited by 41 publications

(45 citation statements)

References 15 publications

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“…In this model, a wrong nucleotide cannot be positioned properly for the nucleophilic attack in the active site of the polymerase, and this slows down the rate of the bond formation significantly. The major role of the base pair geometry in replication has been confirmed recently by Kool and co-workers (19,20), who showed that a thimidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity.…”

mentioning

confidence: 75%

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Dzantiev

Romano

1999

Journal of Biological Chemistry

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DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primertemplates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.It is well established that the presence of DNA adducts in the template strand can impede or block DNA synthesis at a replication fork. Although most bulky adducts inhibit DNA synthesis strongly, some can be bypassed readily in vitro. The well studied carcinogen N-acetyl-2-aminofluorene (AAF) 1 can form both types of adducts in DNA; N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts (dG-C8-AAF) are known to be strong blocks to DNA synthesis, whereas N-(deoxyguanosin-8-yl)-2-aminofluorene adducts (dG-C8-AF) can be bypassed by all polymerases tested (1). The mutagenic consequences of each adduct are also quite distinct. The dG-C8-AAF adduct results in mostly frameshift mutations in bacteria, whereas the dG-C8-AF adduct produces predominantly base substitution mutations (2-4). These different properties are likely to be the result of differences in the structures that these adducts assume in DNA in the active site of the DNA polymerase. Structural and enzymatic studies on duplex DNA molecules have demonstrated that the AF adduct produces less distortion in the DNA helix than the AAF adduct (2, 5). Multidimensional NMR experiments show that the guanine bearing the C8-AAF adduct rotates from anti to syn conformation in the doublestranded DNA helix with the fluorene ring inserted into the helix (base displacement model (6)). This contrasts with the AF adduct, which can adopt interchangeable conformations: in one the fluorene remains outside the helix (outside binding model), whereas the other has the fluorene ring stacked within the helix (5). The ratio of these conformations seems to be dependent on the sequence within which the adduct lies (7). Although it has been assumed that the structural differences between the AAF and AF adducts are responsible for the observed in the...

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mentioning

confidence: 75%

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Dzantiev

Romano

1999

Journal of Biological Chemistry

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“…[116] Ironically, the DNA being synthesized was less stable than the natural DNA by a significant amount. Clearly, however, the DNA polymerase does not sense this destabilization until after the transition REVIEWS E. T. Kool et al Figure 7.…”

Section: Studies With Nonpolar Thymine Analogue Fmentioning

confidence: 99%

“…Interestingly, the DNA polymerase does apparently sense destabilization when multiple F ± A pairs are being synthesized in a row. [116] We suspect that this might arise from the enforced desolvation of the F ± A pairs when they become buried further into the active site.…”

Section: Studies With Nonpolar Thymine Analogue Fmentioning

confidence: 99%

Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication

Kool

Morales

Guckian

2000

Angew. Chem. Int. Ed.

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355

241

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The physical and chemical factors that allow DNA to perform its functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of DNA have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving DNA has been the use of DNA base analogues in which functional groups are added, deleted, blocked, or rearranged. Here we describe a different strategy, in which the polar natural DNA bases are replaced by nonpolar aromatic molecules of the same size and shape. This allows the evaluation of polar interactions (such as hydrogen bonding) with little or no interference from steric effects. We have used these nonpolar nucleoside isosteres as probes of noncovalent interactions such as DNA base pairing and protein - DNA recognition. We have found that, while base-pairing selectivity does depend on Watson - Crick hydrogen bonding in the natural pairs, it is possible to design new bases that pair selectively and stably in the absence of hydrogen bonds. In addition, studies have been carried out with DNA polymerase enzymes to investigate the importance of Watson - Crick hydrogen bonding in enzymatic DNA replication. Surprisingly, this hydrogen bonding is not necessary for efficient enzymatic synthesis of a base pair, and significant levels of selectivity can arise from steric effects alone. These results may have significant impact both on the study of basic biomolecular interactions and in the design of new, functionally active biomolecules.

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“…However, when a run of three adenines was addressed by insertion of analog dFTP, it was found that the second and third adenines presented successively increasing difficulty to the enzyme, and a nearly complete stall was observed after the third apparent insertion of A. 74 Two possible reasons for this observation have been presented: first, the destabilization caused by F-A pairs is likely to be large when they are placed in a run, and this may result in dissociation of primer from template and of polymerase from both. Another possible reason may be poor extension of such pairs, which is typically less favorable than insertion.…”

Section: Prospects For Synthesis Of Dna Strands Without Hydrogen Bondsmentioning

confidence: 99%

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

1998

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Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing

Cited by 41 publications

References 15 publications

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Mimicking the Structure and Function of DNA: Insights into DNA Stability and Replication

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

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