RESUMO
The catalytic function of DNA polymerase ß (pol ß) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol ß can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol ß is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the ß,γ-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group pKa and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol ß. In addition, by utilizing analogue ß,γ-CHX stereoisomers, we identified that the orientation of the ß,γ-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.
Assuntos
DNA Polimerase beta , Conformação Proteica , DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , DNA Polimerase beta/genética , Humanos , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxicitosina/química , Especificidade por Substrato , Modelos Moleculares , Cinética , DNA/metabolismo , DNA/química , Reparo do DNARESUMO
Nucleoside 5'-triphosphate (dNTP) analogues in which the ß,γ-oxygen is mimicked by a CXY group (ß,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-ß,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H2/Pd. This method also affords the ß,γ-CHCl-dTTP (11a, 11b), ß,γ-CHF (12a, 12b), and ß,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in ß,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of ß,γ-CHBr- (33a, 33b), ß,γ-CHCl- (34a, 34b), ß,γ-CHF-dCTP (35a, 35b) were obtained. The ß,γ-CH(CH3)-dATPs (44a, 44b) were obtained using a methyl (R)-(-)-phenylglycinate auxiliary. 31P and 19F NMR Δδ values are correlated with CXY stereochemistry and pKa2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.
Assuntos
DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxicitosina , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
DNA polymerase ß (pol ß) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol ß lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol ß, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol ß within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol ß catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol ß kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.
Assuntos
DNA Polimerase beta/metabolismo , Replicação do DNA/fisiologia , Polipose Adenomatosa do Colo/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Catálise , Neoplasias do Colo/genética , DNA Polimerase beta/química , DNA Polimerase beta/genética , Ligação de Hidrogênio , Cinética , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Especificidade por Substrato , Moldes GenéticosRESUMO
Individual diastereomers of CXY bisphosphonate analogues of dNTPs or NTPs are useful chemical stereoprobes to investigate interactions within the chiral active site environment of enzymes such as polymerases and kinases. We previously reported synthetic access to ß,γ-CHX-dGTPs (X = F or Cl) via a bisphosphonate synthon with an (R)-methyl mandelate auxiliary and have extended this approach to dTTP and dATP analogues. As removal of the chiral auxiliary by (Pd/C) hydrogenolysis is incompatible with the cytosine heterocycle and also with X = Br, we have now designed bisphosphonate synthons using (R)-(+)-α-ethylbenzylamine or methyl (R)-(-)-phenylglycine auxiliaries and equipped with an o-nitrobenzyl ester protecting group allowing photochemical deprotection. These new synthons have made possible the first syntheses of individual dCTP and monobromo-substituted dNTP ß,γ-CHX diastereomers.
RESUMO
We examine the DNA polymerase ß (pol ß) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel ß,γ-CXY-dNTP analogues, including individual ß,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log kpol versus p Ka4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in R and S stereoisomers on the rate-determining step ( kpol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the ß,γ-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site.
Assuntos
DNA Polimerase beta/química , DNA/biossíntese , Catálise , Domínio Catalítico , DNA/química , Humanos , CinéticaRESUMO
The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.
Assuntos
DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Mutação , Neoplasias/genética , DNA Polimerase beta/química , Cinética , Modelos Moleculares , Neoplasias/enzimologia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Nucleotídeos de Timina/metabolismoRESUMO
K289M is a variant of DNA polymerase ß (pol ß) that has previously been identified in colorectal cancer. The expression of this variant leads to a 16-fold increase in mutation frequency at a specific site in vivo and a reduction in fidelity in vitro in a sequence context-specific manner. Previous work shows that this reduction in fidelity results from a decreased level of discrimination against incorrect nucleotide incorporation at the level of polymerization. To probe the transition state of the K289M mutator variant of pol ß, single-turnover kinetic experiments were performed using ß,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pKa4), including a corresponding set of novel ß,γ-CXY dCTP analogues. Surprisingly, we found that the values of the log of the catalytic rate constant (kpol) for correct insertion by K289M, in contrast to those of wild-type pol ß, do not decrease with increased leaving group pKa4 for analogues with pKa4 values of <11. This suggests that one of the relative rate constants differs for the K289M reaction in comparison to that of the wild type (WT). However, a plot of log(kpol) values for incorrect insertion by K289M versus pKa4 reveals a linear correlation with a negative slope, in this respect resembling kpol values for misincorporation by the WT enzyme. We also show that some of these analogues improve the fidelity of K289M. Taken together, our data show that Lys289 critically influences the catalytic pathway of pol ß.