Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10^− 4 to 10^− 5. To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate ? base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K_d. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.     

Functional Importance of Bacteriophage ?29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx₂h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx₂hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.     

Binding of 2′-Amino-2′-Deoxycytidine-5′-Triphosphate to Norovirus Polymerase Induces Rearrangement of the Active Site

Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2′-amino-2′-deoxycytidine-5′-triphosphate have been determined to 1.8 Å resolution. The alteration of the substrate-binding site that is required to accommodate the 2′-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2′-amino-2′-deoxycytidine-5′-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Monogalactosyl diacylglycerol,a replicative DNA polymerase inhibitor,from spinach enhances the anti-cell proliferation effect of gemcitabine in human pancreatic cancer cells

Background

Gemcitabine (GEM) is used to treat various carcinomas and represents an advance in pancreatic cancer treatment. In the screening for DNA polymerase (pol) inhibitors, a glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), was isolated from spinach.

Methods

Phosphorylated GEM derivatives were chemically synthesized. In vitro pol assay was performed according to our established methods. Cell viability was measured using MTT assay.

Results

Phosphorylated GEMs inhibition of mammalian pol activities assessed, with the order of their effect ranked as: GEM-5′-triphosphate (GEM-TP) > GEM-5′-diphosphate > GEM-5′-monophosphate > GEM. GEM suppressed growth in the human pancreatic cancer cell lines BxPC-3, MIAPaCa2 and PANC-1 although phosphorylated GEMs showed no effect. MGDG suppressed growth in these cell lines based on its selective inhibition of replicative pol species. Kinetic analysis showed that GEM-TP was a competitive inhibitor of pol α activity with nucleotide substrates, and MGDG was a noncompetitive inhibitor with nucleotide substrates. GEM combined with MGDG treatments revealed synergistic effects on the inhibition of DNA replicative pols α and γ activities compared with GEM or MGDG alone. In cell growth suppression by GEM, pre-addition of MGDG significantly enhanced cell proliferation suppression, and the combination of these compounds was found to induce apoptosis. In contrast, GEM-treated cells followed by MGDG addition did not influence cell growth.

Conclusions

GEM/MGDG enhanced the growth suppression of cells based on the inhibition of pol activities.

General significance

Spinach MGDG has great potential for development as an anticancer food compound and could be an effective clinical anticancer chemotherapy in combination with GEM.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+

2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn^2 +, a new conserved motif, (G⁴³YNG⁴⁶), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.     

Synthesis and biological evaluation of fatty acyl ester derivatives of 2',3'-didehydro-2',3'-dideoxythymidine

A number of 5′-O-fatty acyl derivatives of 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5′-O-12-azidododecanoyl derivative of d4T (2), displaying EC₅₀ = 3.1-22.4 μM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through β-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine.     

Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2′-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2′-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2′-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.     

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 μM. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2′-deoxyguanosine and 1,N²-etheno-2′-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells.     

Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases

Chronic inflammation involving constant generation of nitric oxide (NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2′-deoxyinosine (dI) and 2′-deoxyxanthosine are primarily formed by NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol α was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol η and pol κΔC (a truncated form of pol κ) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol α, pol η, and pol κΔC, promoted preferential incorporation of 2′-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2′-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol α, pol η, and pol κΔC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A → G transition. This NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: A simultaneous protein binding assay

The adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate contents of microliter quantities of urine can be determined simultaneously by combining individual protein binding assays for the two nucleotides. ³²P-labeled adenosine 3′,5′-monophosphate is bound to a protein from bovine skeletal muscle, while a lobster muscle protein preparation is utilized for binding of ³H-labeled guanosine 3′,5′-monophosphate.