NMR Structure of a Gemcitabine-Substituted Model Okazaki Fragment

Konerding, David E.; James, Thomas Leroy; Trump, Eric; Soto, Ana M.; Marky, Luis A.; Gmeiner, William H.

doi:10.1021/bi015678l

Cited by 23 publications

(45 citation statements)

References 29 publications

(45 reference statements)

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“…Similarly, dFd-CMP also decreased the binding affinity of the next 2 downstream nucleotides by 6 -10-fold before recovering to normal. The predicted decrease in the structural integrity of the DNA duplex caused by the presence of a dFdCMP moiety with a 3Ј-endo pucker is supported by the fact that the melting temperature of the 12-bp Okazaki fragment is lowered by 4.3°C in the presence of an internal dFdCMP (44). Such a noncanonical conformation of dFdCMP in a template should also affect the positioning of an incoming dNTP for in-line attack by the primer's 3Ј-OH group during phosphodiester bond formation.…”

Section: Discussionmentioning

confidence: 94%

“…Following dFdCTP incorporation, the DNA primer terminated with 3Ј-dFdCMP is likely to adopt the 3Ј-endo pucker conformation as observed in the solution structures of gemcitabine embedded in DNA (44). The electron density of its 3Ј-OH group should be lowered by the electron-withdrawing fluorine group, thereby rendering the 3Ј-OH group less nucleophilic.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase

Fowler

Brown

Johnson

et al. 2008

Journal of Biological Chemistry

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Gemcitabine, 2-deoxy-2,2-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase ␥. Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase ␥. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214-and 7-fold, respectively. Moreover, the primer 3-dFdCMP was excised with a 50-fold slower rate than the matched 3-dCMP. When dFdC was encountered as a template base, DNA polymerase ␥ paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.Many nucleoside analogs are potent anti-cancer and antiviral drug compounds. Among 15 Food and Drug Administration-approved nucleoside analogs, gemcitabine or 2Ј-deoxy-2Ј,2Ј-difluorocytidine (dFdC, 4 supplemental Fig. 1) is an anticancer drug that is clinically used for the treatment of nonsmall cell lung cancer (1), pancreatic cancer (2), metastatic breast cancer (3), and ovarian cancer (4). It has also shown promising efficacy for the treatment of other solid tumors and hematological malignancies (5-12) suggesting more widespread use in the future. In addition to its use as a monotherapy, gemcitabine is often most effective when used as part of a combination therapy, frequently with platinum-based and topoisomerase-targeted chemotherapeutic agents (13-15).Gemcitabine is administered in the form of a biologically inactive prodrug that first permeates the cellular membrane by facilitated diffusion (16, 17) almost exclusively via the human equilibrative nucleoside transporter number 1 (17, 18). Following transport, dFdC is metabolized to the biologically active monophosphorylated form (dFdCMP) by deoxycytidine kinase, which is the rate-limiting step during the activation of gemcitabine (19). Subsequently, dFdCMP is further phosphorylated to form the cytotoxic metabolites gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) by cellular kinases. It has been shown that dFdCTP com...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase

Fowler

Brown

Johnson

et al. 2008

Journal of Biological Chemistry

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“…Studies have shown that the arabinose sugar moiety in AraC and the difluoro group on the 2Ј-position of the sugar moiety of dFdC alter the DNA structure, inhibiting DNA polymerases (11,12). The cytotoxicity of AraC or dFdC is proportional to the amount of the analog incorporated into DNA (6 -8).…”

mentioning

confidence: 99%

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

García‐Díaz

Murray

Kunkel

et al. 2010

Journal of Biological Chemistry

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The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase (Pol ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol . Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol could modulate the cytotoxic effect of AraC.Nucleoside analogs are an important class of compounds that are clinically used for anticancer and antiviral treatments (1). For example, 3TC, 3 AZT, and D4T have been used for antiviral treatments (1). AraC ( Fig. 1) is used to treat leukemia (2), and dFdC ( Fig. 1) is used to treat various types of cancer, such as non-small cell lung cancer (3), breast cancer (4), and pancreatic cancer (5). The main mechanism of action of nucleoside analogs is to terminate the DNA elongation process after their incorporation into viral or cellular DNA (6 -8). For example, due to the lack of a 3Ј-hydroxyl group on their sugar moieties, the incorporation of the antiviral compounds 3TC, AZT, and D4T terminates human immunodeficiency virus DNA replication immediately (9).Although both AraC and gemcitabine have a 3Ј-hydroxyl group on their sugar moieties, the incorporation of either compound has been shown to terminate the DNA elongation processes and result in DNA fragmentation (7,10). Studies have shown that the arabinose sugar moiety in AraC and the difluoro group on the 2Ј-position of the sugar moiety of dFdC alter the DNA structure, inhibiting DNA polymerases (11,12). The cytotoxicity of AraC or dFdC is proportional to the amount of the analog incorporated into DNA (6 -8). Therefore, the cellular enzymes that are involved in activating these nucleoside analogs and the DNA polymerases that introduce them into DNA play a critical role in determining their cytotoxic activity.Human DNA polymerase (Pol ) is an exonuclease-deficient, 575-amino acid DNA polymerase that belongs to the X-family (13). It contains a N-terminal BRCT domain that is important for protein partnerships, a proline/serine-rich region that has been suggested to be a target for post-translational modification, and a C-terminal 39-kDa polymerase domain (14). The 39-kDa domain, which is structurally well characterized (15, 16) contains the fingers, palm, and thumb subdomains typical of DNA polymerases (17). It also contains an 8-kDa subdomain with dRP lyase activity (14) that contributes to base excision repair (BER) in vitro. Studies in Pol ␤-deficient mouse embryonic fibroblast extracts indicate that Pol also performs BER in vivo (18,19). Moreover, in a confocal microscopy study, Pol reloca...

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“…Several mechanisms have been reported, including enzymes involved in modulating the nucleotide pool (28), or DNA exonuclease that can remove these analogs from the 3 0 termini of DNA and therefore diminish their activities (27). Structural and mechanistic studies showed that the arabinose sugar moiety of AraC and the di-fluoro group on the 2 0 position of the sugar moiety of dFdC alter the DNA structure, which significantly reduced the extension efficiency for replicative DNA polymerases (41,54). In comparison, pol Z efficiently extends from either AraC or gemcitabine at the 3 0 termini of DNA (22).…”

Section: Dna Polymerase Eta and Anticancer Agentsmentioning

confidence: 99%

DNA Polymerase Eta and Chemotherapeutic Agents

Chou

2011

Antioxidants & Redox Signaling

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The discovery of human DNA polymerase eta (pol Z) has a major impact on the fields of DNA replication=repair fields. Since the discovery of human pol Z, a number of new DNA polymerases with the ability to bypass various DNA lesions have been discovered. Among these polymerases, pol Z is the most extensively studied lesion bypass polymerase with a defined major biological function, that is, to replicate across the cyclobutane pyrimidine dimers introduced by UV irradiation. Cyclobutane pyrimidine dimer is a major DNA lesion that causes distortion of DNA structure and block the replicative DNA polymerases during DNA replication process. Genetic defects in the pol Z gene, Rad30, results in a disease called xeroderma pigmentosum variant. This review focuses on the overall properties of pol Z and the mechanism that involved in regulating its activity in cells. In addition, the role of pol Z in the action of DNA-targeting anticancer compounds is also discussed.

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NMR Structure of a Gemcitabine-Substituted Model Okazaki Fragment

Cited by 23 publications

References 29 publications

Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase

Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

DNA Polymerase Eta and Chemotherapeutic Agents

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