Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity

Devadoss, Babho; Berdis, Anthony J.

doi:10.2174/187231307781662215

Cited by 4 publications

(4 citation statements)

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“…Because only dNaMTP or d5SICSTP exist in the reaction without any other canonical nucleotides, they will be the only nucleotides inserted. Literature precedence has demonstrated that Klenow fragment of DNA polymerase I deficient in exonuclease activity (Kf exo − ) has the potential to react at a gap site in a duplex, as well as being capable of inserting non-natural nucleotides 41 42 ; hence, Kf exo − was selected for study of inserting either dNaMTP or d5SICSTP opposite a template dG. On the basis of PAGE analysis ( Figs 3b and 4b ), Kf exo − with either of the marker triphosphates added to the reaction from step II could insert opposite a template dG to furnish the nicked duplex in a 1-h incubation with a nearly quantitative yield ( Figs 3c and 4c ).…”

Section: Resultsmentioning

confidence: 99%

Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

et al. 2015

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Damage to the genome is implicated in the progression of cancer and stress-induced diseases. DNA lesions exist in low levels, and cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied and sequenced. Critical for this method is installation of either the dNaM or d5SICS nucleotides at the lesion site after processing via the base excision repair process. These marker nucleotides constitute an unnatural base pair, allowing large quantities of marked DNA to be made by PCR amplification. Sanger sequencing confirms the potential for this method to locate lesions by marking, amplifying and sequencing a lesion in the KRAS gene. Detection using the α-hemolysin nanopore is also developed to analyse the markers in individual DNA strands with the potential to identify multiple lesions per strand.

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Section: Resultsmentioning

confidence: 99%

Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

et al. 2015

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“…The classical approach to develop chemotherapeutic agents targeting DNA pols was to investigate sugar or nucleobase modiﬁcations in the nucleoside precursor, based on a SAR method [ 77 , 78 , 79 , 80 ]. A great advantage for the development of new effective NAs comes from the analysis of the crystal resolutions of DNA pols in complex with different NAs.…”

Section: Nucleoside Analogs As Dna Pol β and λ Inhibitorsmentioning

confidence: 99%

“…Surely, the lack of crystallographic resolutions of these DNA pols in complexes with other clinically approved NAs represents an obstacle for the design of new effective molecules. On the other hand, thanks to many crystallographic resolutions as well as pre-steady state kinetic and SAR analysis of DNA pol β and λ with natural or unnatural substrates [ 77 , 78 , 79 , 80 , 84 , 85 , 86 , 80 , 84 ], the mechanistic details governing substrate binding and catalysis are known for these two pols to a much deeper level than for the majority of other mammalian pols (with the exception maybe of the translesion enzymes DNA pols η, ι and κ). Such an improved understanding can provide useful information for the design of new effective NAs.…”

Section: Nucleoside Analogs As Dna Pol β and λ Inhibitorsmentioning

confidence: 99%

Exploiting the Nucleotide Substrate Specificity of Repair DNA Polymerases To Develop Novel Anticancer Agents

et al. 2011

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The genome is constantly exposed to mutations that can originate during replication or as a result of the action of both endogenous and/or exogenous damaging agents [such as reactive oxygen species (ROS), UV light, genotoxic environmental compounds, etc.]. Cells have developed a set of specialized mechanisms to counteract this mutational burden. Many cancer cells have defects in one or more DNA repair pathways, hence they rely on a narrower set of specialized DNA repair mechanisms than normal cells. Inhibiting one of these pathways in the context of an already DNA repair-deficient genetic background, will be more toxic to cancer cells than to normal cells, a concept recently exploited in cancer chemotherapy by the synthetic lethality approach. Essential to all DNA repair pathways are the DNA pols. Thus, these enzymes are being regarded as attractive targets for the development of specific inhibitors of DNA repair in cancer cells. In this review we examine the current state-of-the-art in the development of nucleotide analogs as inhibitors of repair DNA polymerases.

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“…Despite the nontemplating nature of an abasic site, most DNA polymerases preferentially incorporate dATP opposite the lesion . We applied this information in rationally designing a series of 5-substituted indolyl-2′-deoxyriboside triphosphates that mimic the core structure of dATP (Figure ) (reviewed in refs and ). In vitro kinetic analyses reveal that analogues containing significant π-electron density such as 5-NITP, 5-Ph-ITP, and 5-CE-ITP are incorporated opposite the abasic site with high catalytic efficiencies of 10 7 M −1 s −1 .…”

Section: Dna Replication As a Therapeutic Targetmentioning

confidence: 99%

DNA Polymerases as Therapeutic Targets

Berdis

2008

Biochemistry

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127

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Numerous pathological states, including cancer, autoimmune diseases, and viral/bacterial infections, are often attributed to uncontrollable DNA replication. Inhibiting this essential biological process provides an obvious therapeutic target against these diseases. A logical target is the DNA polymerase, the enzyme responsible for catalyzing the addition of mononucleotides to a growing polymer using a DNA or RNA template as a guide for directing each incorporation event. This review provides a summary of therapeutic agents that target polymerase activity. A discussion of the biological function and mechanism of polymerases is first provided to illustrate the strategy for therapeutic intervention as well as the rational design of various nucleoside analogues that inhibit various polymerases associated with viral infections and cancer. The development of nucleoside and non-nucleoside inhibitors as antiviral agents is discussed with particular emphasis on their mechanism of action, structure-activity relationships, toxicity, and mechanism of resistance. In addition, commonly used anticancer agents are described to illustrate the similarities and differences associated with various nucleoside analogues as therapeutic agents. Finally, new therapeutic approaches that include the inhibition of selective polymerases involved in DNA repair and/or translesion DNA synthesis as anticancer agents are discussed.

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Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity

Cited by 4 publications

References 0 publications

Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

Exploiting the Nucleotide Substrate Specificity of Repair DNA Polymerases To Develop Novel Anticancer Agents

DNA Polymerases as Therapeutic Targets

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