Despite the fact that the number of publications associated with the keyword 'enzyme' increases every year, the precise origin of enzyme catalysis has remained unresolved. Because of sustained intensive research efforts from an increasing number of laboratories, detailed information regarding the physics, chemistry and kinetics of enzymes is accumulating rapidly. The growing body of data contains many examples of kinetic behavior that are incompatible with a static view of enzyme catalysis. As a result, numerous laboratories are approaching the consensus that protein motion plays an essential role in enzyme catalysis. A model that incorporates nuclear quantum tunneling together with two classes of protein motion--termed conformational sampling (pre-organization) and reorganization--is recommended as a means of understanding the large body of data for enzyme-catalyzed hydrogen transfers. It should also serve as a vehicle for future efforts in the development of potent enzyme inhibitors and the de novo design of all classifications of enzymes.

ARID1A (the AT-rich interaction domain 1A, also known as BAF250a) is one of the most commonly mutated genes in cancer1,2. The majority of ARID1A mutations are inactivating mutations and lead to loss of ARID1A expression3, which makes ARID1A a poor therapeutic target. Therefore, it is of clinical importance to identify molecular consequences of ARID1A deficiency that create therapeutic vulnerabilities in ARIDIA-mutant tumors. In a proteomic screen, we found that ARID1A interacts with mismatch repair (MMR) protein MSH2. ARID1A recruited MSH2 to chromatin during DNA replication and promoted MMR. Conversely, ARID1A inactivation compromised MMR and increased mutagenesis. ARID1A deficiency correlated with microsatellite instability genomic signature and a predominant C>T mutation pattern and increased mutation load across multiple human cancer types. Tumors formed by an ARID1A-deficient ovarian cancer cell line in syngeneic mice displayed increased mutation load, elevated numbers of tumor-infiltrating lymphocytes, and PD-L1 expression. Notably, treatment with anti-PD-L1 antibody reduced tumor burden and prolonged survival of mice bearing ARIDIA-deficient but not ARID1A-wild-type ovarian tumors. Together, these results suggest ARID1A deficiency contributes to impaired MMR and mutator phenotype in cancer, and may cooperate with immune checkpoint blockade therapy.

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The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.

consults and holds stock in Ideaya, and cofounded and holds stock in Cedilla Therapeutics. G.G. receives research funding from IBM and Pharmacyclics and is an inventor on multiple patent applications related to bioinformatic tools, including applications related to MuTect, ABSOLUTE, MSMuTect, MSMutSig and MSIClass. Y.E.M. is an inventor on patent applications related to the bioinformatic tools, MSMuTect, MSMutSig and MSIClass. The Broad Institute filed a US patent application related to the target described in this manuscript.

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.

A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10 13 s −1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.A complete understanding of the origins of the remarkable rate acceleration and specificity achieved by enzymes remains elusive. Increasingly, studies on the fundamental basis for enzymatic catalysis have focused on the requirement for a range of protein motions in order to achieve efficient enzyme catalysis. Among these, evidence for a class of motion termed conformational sampling (or preorganization) has begun to accumulate recently (cf. refs. 1-4). The impact of such motions can be seen either in the formation of an enzyme-substrate complex or in its subsequent conversion to product.For example, a recent study of adenylate kinase implicates a temperature-dependent preequilibrium among conformers that are either "competent" or "incompetent" toward ligand binding (5). In the context of catalysis, studies of single-enzyme molecules indicate multiple, slowly interconverting conformers that lead to product with different rate constants (6-8). Extensive work on dihydrofolate reductase has shown the role of slow loop closures that occur along the reaction coordinate with the same (millsecond) time constants as catalysis (9, 10).Although computational studies also suggest a role for rapid conformational sampling in enzyme reactions (11, 12), demonstrating the direct link of these motions to catalysis is quite challenging. In this study, we use the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) as a system to examine the presence of rapidly interconverting protein substates during catalysis. The ht-ADH catalyzes the transfer of a hydride equivalent from an alcohol donor to an NAD þ cofactor, and previous studies have shown that ht-ADH carries out its reaction via quantum t...