A pinboard by
Khadijeh Alnajjar

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.


Catalytic effects of mutations of distant protein residues in human DNA polymerase β: theory and experiment.

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

Fluorescence resonance energy transfer studies of DNA polymerase β: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection.

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

A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase Beta Cancer-Associated Variant.

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