Postdoctoral associate at Yale University. Biochemistry roles!
A biochemical approach to understand how sequence-context affects errors made by polymerases
Certain genes in our genome are prone to high mutation frequency, creating signature hotspots, which are characteristic of certain cancers. Many of these hotspots have been identified, however, no one has been able to understand why these specific hotspots occur. The focus of my work is to test the hypothesis that certain sequences affect the reaction mechanism of DNA polymerases, allowing for increased error frequency. Specifically, we were interested in hotspots occurring the APC gene, which is a tumor suppressor gene mutated in 80% of colorectal cancer patients. DNA polymerases are essential because they are involved in faithfully replicating DNA while making minimal errors. Because DNA is frequently damaged by exposure to damaging agents such as UV light or reactive oxygen species produced from cellular metabolism along with other sources, polymerases become essential for our survival. The base excision repair pathway repairs DNA damage and the polymerase responsible for faithfully incorporating correct nucleotides is DNA polymerase beta. Mutations in polymerase beta can lead to increased mutation rates in certain genes, which can lead to carcinogenesis. Ongoing studies suggest that a mutation in polymerase beta increases mistakes made by the enzyme in a sequence-context specific manner, which changes the reaction mechanism of the enzyme. This conclusion was made by studying the rate-determining step (RDS) of the mutant enzyme (chemical vs. conformational) in the presence of various sequences. Our results suggest that in the presence of a sequence reoccurring in the APC gene, the RDS of a polymerase beta mutant becomes conformational, allowing for increased error frequency. However, in the presence of a control sequence, the reaction mechanism is equivalent to the wild-type mechanism, having a RDS that is chemical. These results will be essential to understand the signature hotspots occurring in 80% of colorectal cancer patients.
Abstract: DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.
Pub.: 10 Sep '03, Pinned: 30 Jun '17
Abstract: Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.
Pub.: 30 Nov '05, Pinned: 30 Jun '17
Abstract: Previous small scale sequencing studies have indicated that DNA polymerase β (pol β) variants are present on average in 30% of human tumors of varying tissue origin. Many of these variants have been shown to have aberrant enzyme function in vitro and to induce cellular transformation and/or genomic instability in vivo, suggesting that their presence is associated with tumorigenesis or its progression. In this study, the human POLB gene was sequenced in a collection of 134 human colorectal tumors and was found to contain coding region mutations in 40% of the samples. The variants map to many different sites of the pol β protein and are not clustered. Many variants are nonsynonymous amino acid substitutions predicted to affect enzyme function. A subset of these variants was found to have reduced enzyme activity in vitro and failed to fully rescue pol β-deficient cells from methylmethane sulfonate-induced cytotoxicity. Tumors harboring variants with reduced enzyme activity may have compromised base excision repair function, as evidenced by our methylmethane sulfonate sensitivity studies. Such compromised base excision repair may drive tumorigenesis by leading to an increase in mutagenesis or genomic instability.
Pub.: 12 May '12, Pinned: 30 Jun '17
Abstract: We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol β mutants.
Pub.: 28 Sep '12, Pinned: 30 Jun '17
Abstract: During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.
Pub.: 26 Apr '14, Pinned: 30 Jun '17
Abstract: K289M is a variant of DNA polymerase β (pol β) that was previously identified in colorectal cancer. The expression of this variant leads to a 16-fold increase in mutation frequency at a specific site <i>in vivo</i> and a reduction in fidelity <i>in vitro</i> in a sequence context-specific manner. Previous work shows that this reduction in fidelity results from decreased discrimination against incorrect nucleotide incorporation at the level of polymerization. To probe the transition state of the K289M mutator variant of pol β, single turnover kinetics experiments were performed using β,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (p<i>K</i><sub>a4</sub>), including a corresponding set of novel β,γ-CXY dCTP analogues. Surprisingly, we found that the log of the catalytic rate constants (<i>k</i><sub>pol</sub>) for <i>correct</i> insertion by K289M, in contrast to those of wild-type pol β, do not decrease with increased leaving group p<i>K</i><sub>a4</sub> for analogues with p<i>K</i><sub>a4</sub> < 11. This suggests that one of the relative rate constants differs for the K289M reaction in comparison to that of WT. However, a plot of log(<i>k</i><sub>pol</sub>) values for <i>incorrect</i> insertion by K289M vs. p<i>K</i><sub>a4</sub reveal a linear correlation with a negative slope, in this respect resembling <i>k</i><sub>pol</sub> values for misincorporation by wild-type 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 DNA pol β.
Pub.: 23 Mar '17, Pinned: 30 Jun '17
Abstract: With the formidable growth in genetic information, it has become essential to identify and characterize mutations in macromolecules in order to not only predict contributions to disease processes but also to guide the design of therapeutic strategies. While mutations of certain residues have a predictable phenotype based on their chemical nature and known structural position, many types of mutations evade prediction based on current information. Described in this work are the crystal structures of two cancer variants located in the palm domain of DNA polymerase beta (pol β), S229L and G231D, whose biological phenotype was not readily linked to a predictable structural implication. Structural results demonstrate that the mutations elicit their effect through subtle influences on secondary interactions with a residue neighboring the active site. Residues 229 and 231 are 7.5 and 12.5 Å, respectively, from the nearest active site residue, with a β-strand in between. A residue on this intervening strand, M236, appears to transmit fine structural perturbations to the catalytic metal-coordinating residue D256, affecting its conformational stability.
Pub.: 14 Apr '17, Pinned: 30 Jun '17