Collapsed State Mediates the Low Fidelity of the DNA Polymerase ß I260 Mutant.

DNA polymerase ß (Pol ß) fills single nucleotide gaps during base excision repair. Deficiencies in Pol ß can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol ß, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Förster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol ß by creating a "collapsed" state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the "fingers closing" conformational change for polymerase fidelity and accurate DNA synthesis.

Modifying the Basicity of the dNTP Leaving Group Modulates Precatalytic Conformational Changes of DNA Polymerase ß.

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.

The role of cysteines in the structure and function of OGG1.

8-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA modification. OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress-response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation. Due to the integral role of OGG1 in maintaining cellular homeostasis under oxidative stress, it is important to understand the effect of oxidants on OGG1 and the role of cysteines in its structure and function. In this study, we investigate the role of the cysteine residues in the function of OGG1 by mutating and characterizing each cysteine residue. Our results indicate that the cysteines in OGG1 fall into four functional categories: those that are necessary for (1) glycosylase activity (C146 and C255), (2) lyase activity (C140S, C163, C241, and C253), and (3) structural stability (C253) and (4) those with no known function (C28 and C75). These results suggest that under conditions of oxidative stress, cysteine can be targeted for modifications, thus altering the response of OGG1 and affecting its downstream cellular functions.

Mutational signature profiling classifies subtypes of clinically different mismatch-repair-deficient tumours with a differential immunogenic response potential.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

Cost-Effectiveness and Economic Burden Analyses on All First-Line Treatments of Chronic Lymphocytic Leukemia.

OBJECTIVES: Several chemoimmunotherapy and targeted treatment regimens are approved as front-line therapies in chronic lymphocytic leukemia. We estimated for the 10-year cost-effectiveness of these treatment regimens and the economic burden of following the estimated risk-stratified 21 040 patients with chronic lymphocytic leukemia diagnosed in 2020 for 10 years. METHODS: A Markov model with 7 exclusive health states was specified over a 10-year time horizon. Treatment effectiveness inputs were obtained from a novel network meta-analysis on the progression-free survival, overall survival curves, and time to next treatment. Costs and utilities inputs were included for each health state for each treatment and discounted at 3.0%/year. Life-years (LYs) and quality-adjusted LYs (QALYs) for each treatment were determined. Using the lowest cost regimen as reference, the incremental cost-effectiveness ratio (ICER) and incremental cost-utility ratio (ICUR) were estimated. The 10-year per-patient cost was determined by risk status and by initial treatment. RESULTS: Venetoclax-plus-obinutuzumab was the lowest cost regimen, hence the reference. Superior in effectiveness to all chemoimmunotherapies, it was cost saving. With the highest effectiveness gains at 6.26 LYs and 5.01 QALYs and despite being the most expensive regimen ($1 298 638 per patient), acalabrutinib-plus-obinutuzumab yielded the best ICER ($409 343/LY gained) and ICUR ($501 236/QALY gained). The remaining ICERs of targeted therapies ranged from $512 101/LY gained to $793 236/LY gained and the ICURs from $579 737/QALY gained to $869 300/QALY gained. The 10-year postdiagnosis low/high (venetoclax-plus-obinutuzumab/acalabrutinib-plus-obinutuzumab) economic burden ranges were $42 690 to $98 665 for low-risk, $141 339 to $326 660 for intermediate-risk, and $273 650 to $632 453 for high-risk patients. CONCLUSIONS: Compared with venetoclax-plus-obinutuzumab, chemoimmunotherapies are associated with less health benefits at higher cost. The targeted therapies achieve greater benefits at higher cost.

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase ß.

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase ß (pol ß) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol ß has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol ß with r8-oxo-GTP, we demonstrate impaired pol ß closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase ß-DNA complexes.

Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Survival trends in chronic lymphocytic leukemia across treatment eras: US SEER database analysis (1985-2017).

In this population-based study, we used the SEER database (1985-2015) to examine survival outcomes in chronic lymphocytic leukemia (CLL) patients followed up to the era of advanced treatments including targeted therapies. Data were extracted for patients 15 years or older with a primary diagnosis of CLL. A period analysis was performed to estimate 5- and 10-year relative survival rates for patients diagnosed during different calendar periods from 1985 to 2015. A mixture cure model was used to examine long-term survivors' proportions among patients diagnosed in 1985-2015 and for two cohorts diagnosed in 2000-2003, followed up to 2012 and 2004-2007, and followed up to 2015. Cox proportional hazard modeling was used for the two cohorts to estimate hazard ratios (HRs) of death adjusted for gender and age. The 5-year and 10-year age-adjusted relative survival rate ranged between 73.7 and 89.4% and from 51.6% to "not reached," respectively, for calendar periods of 1985-1989 to 2010-2014. The long-term survivor proportions varied by age and gender from 0 to 59%. The HRs (95%CI) for the 2004-2007 cohort in comparison to the 2000-2003 cohort were 0.58 (0.43-0.78), 0.58 (0.48-0.70), 0.57 (0.49-0.0.67), 0.68 (0.54-0.85), and 0.83 (0.68-1.02) for the age categories of 45-54, 55-64, 65-74, 75-84, and ≥ 85 years, respectively. Overall, relative survival improved significantly for CLL patients diagnosed between 1985 and 2015. These improvements were markedly better following the introduction of targeted therapies.

A pre-catalytic non-covalent step governs DNA polymerase ß fidelity.

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.

Revealing an Internal Stabilization Deficiency in the DNA Polymerase ß K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography.

The human DNA polymerase (pol) ß cancer variant K289M has altered polymerase activity in vitro, and the structure of wild-type pol ß reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF2 group substitution for the ß-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol ß is not altered in the presence of the dGTP CF2 analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol ß and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.

The double-edged sword of cancer mutations: exploiting neoepitopes for the fight against cancer.

Defects in DNA repair have been linked to the accumulation of somatic mutations in tumours. These mutations can promote oncogenesis; however, recent developments have indicated that they may also lead to a targeted immune response against the tumour. This response is initiated by the development of new antigenic epitopes (neoepitopes) arising from mutations in protein-coding genes that are processed and then presented on the surface of tumour cells. These neoepitopes are unique to the tumour, thus enabling lymphocytes to launch an immune response against the cancer cells. Immunotherapies, such as checkpoint inhibitors (CPIs) and tumour-derived vaccines, have been shown to enhance the immunogenic response to cancers and have led to complete remission in some cancer patients. There are tumours that are not responsive to immunotherapy or conventional tumour therapeutics; therefore, there is a push for new treatments to combat these unresponsive cancers. Recently, combinatorial treatments have been developed to further utilise the immune system in the fight against cancer. These treatments have the potential to exploit the defects in DNA repair by inducing more DNA damage and mutations. This can potentially lead to the expression of high levels of neoepitopes on the surface of tumour cells that will stimulate an immunological response. Overall, exploiting DNA repair defects in tumours may provide an edge in this long fight against cancer.

DNA polymerase beta participates in DNA End-joining.

DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol µ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol ß), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol ß, fewer small deletions are observed. In addition, depletion of Pol ß results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol ß in functions in aNHEJ and provide mechanistic insight into its role in this process.

I260Q DNA polymerase ß highlights precatalytic conformational rearrangements critical for fidelity.

DNA polymerase ß (pol ß) fills single nucleotide gaps in DNA during base excision repair and non-homologous end-joining. Pol ß must select the correct nucleotide from among a pool of four nucleotides with similar structures and properties in order to maintain genomic stability during DNA repair. Here, we use a combination of X-ray crystallography, fluorescence resonance energy transfer and nuclear magnetic resonance to show that pol ß's ability to access the appropriate conformations both before and upon binding to nucleotide substrates is integral to its fidelity. Importantly, we also demonstrate that the inability of the I260Q mutator variant of pol ß to properly navigate this conformational landscape results in error-prone DNA synthesis. Our work reveals that precatalytic conformational rearrangements themselves are an important underlying mechanism of substrate selection by DNA pol ß.

The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase ß.

DNA polymerase ß (Pol ß) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol ß incorporates a nucleotide into a single-gapped DNA substrate. Pol ß may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5'-modified downstream DNA. We have recently shown that Pol ß fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol ß undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol ß with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5'-phosphate group has a more significant effect. The absence of the 5'-phosphate decreases the DNA binding affinity of Pol ß and results in a conformationally more open binary complex. Moreover, upon addition of the correct nucleotide in the absence of 5'-phosphate, a slow fingers closing step is observed. Interestingly, either increasing the gap size or removing the 5'-phosphate group results in loss of the noncovalent step. Together, these results suggest that the character of the DNA substrate impacts the nature and rates of pre-catalytic conformational changes of Pol ß. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol ß.

The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype.

The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

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.

Heterozygosity for a hypomorphic Polß mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polß, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

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

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.

Remote Mutations Induce Functional Changes in Active Site Residues of Human DNA Polymerase ß.

With the formidable growth in the volume of genetic information, it has become essential to identify and characterize mutations in macromolecules not only to 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 ß (pol ß), 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 ß-strand between them. A residue on this intervening strand, M236, appears to transmit fine structural perturbations to the catalytic metal-coordinating residue D256, affecting its conformational stability.

DNA Polymerase ß Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ß-γ Bridging Group of the Incoming dNTP.

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.