SEARCH · Search bioRxiv

Results for “biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

bioRxiv2019

Inducible degron-dependent depletion of the RNA polymerase I associated factor PAF53 demonstrates it is essential for cell growth and allows for the analysis of functional domains

Our knowledge of the mechanism of rDNA transcription has benefitted from the combined application of genetic techniques in yeast, and progress on the biochemistry of the various components of yeast rDNA transcription. Nomuras laboratory derived a system in yeast for screening for mutants essential for ribosome biogenesis. Such systems have allowed investigators to not only determine if a gene was essential, but to analyze domains of the proteins for different functions in rDNA transcription in vivo. However, because there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an inducible degron that allows us to combine a \"genetics-like\" approach to studying mammalian rDNA transcription with biochemistry. Using this system, we show that the mammalian homologue of yeast A49, PAF53, is required for rDNA transcription and mitotic growth. Further, we have been able to study the domains of the protein required for activity. We have found that while the C-terminal, DNA-binding domain (tWH) was necessary for complete function, the heterodimerization and linker domains were also essential. Analysis of the linker identified a putative DNA-binding domain. We have confirmed that the helix-turn-helix (HTH) of the linker constitutes a second DNA-binding domain within PAF53 and that the HTH is essential for PAF53 function.

molecular biology↗

bioRxiv2026

Metabolites Shift Equilibria of Biomolecular Condensates

Biomolecular condensates compartmentalize biochemistry in living cells. While in vitro models of condensates involve only a few components, the cytoplasm is a complex mixture with thousands of components, including many small molecules. While many macromolecular drivers of phase separation have been revealed, the contributions from small molecules have received little attention. To quantify the impact of solutes on biomolecular condensates, we introduce susceptibility, a dimensionless descriptor of condensate response to solute perturbations. We measured how three model condensates, assembled by distinct cohesive mechanisms, respond to diverse solutes including amino acids and nucleotides. Generically, solutes shift condensate phase equilibria, with susceptibilities spanning over four orders of magnitude. These values reflect underlying molecular interactions, consistent with theoretical descriptions including Flory-Huggins and polyphasic linkages. As one example of the predictive power of susceptibility, we exploit enzymatic activity to induce condensation and modulate material properties. Our work establishes susceptibility as an indicator of the sensitivity of biomolecular condensates to solutes, with implications for cell physiology and therapeutic design. SIGNIFICANCE STATEMENTCells compartmentalize biochemistry using biomolecular condensates formed through phase separation. Although cells contain thousands of small molecules, little is known about their influence on condensation. In such complex mixtures, mapping full phase diagrams is infeasible. Alternatively, we introduce susceptibility to characterize system response around a working composition. Using three distinct model condensates and over a dozen solutes, we observe susceptibilities varying over four orders of magnitude. We provide a general thermodynamic framework that clarifies the driving forces behind these responses, rationalizing their magnitude and their dependence on location in the phase diagram. Our work provides a framework for understanding and harnessing solutes to regulate biomolecular condensation, with implications for cell physiology and therapeutic design.

biophysics↗

bioRxiv2026

ILK binding to β1 Integrin is indirectly mediated by Kindlin-2

Integrin-linked kinase (ILK) and kindlin-2 (K2) are key components of focal adhesions (FAs) that regulate cell-matrix adhesion and integrin signaling. Both proteins directly bind each other, but how they influence each others localization to FAs and binding to integrins remains a subject of ongoing debate. Here, we establish a sensitive workflow to study protein-protein interactions in cells by combining methods from biochemistry, cell biology and super-resolution microscopy. Together with an analytical framework this approach allowed us to distinguish direct from indirect molecular interactions and construct detailed interaction networks. Disrupting the ILK-K2 interaction reduced ILK localization to FAs and compromised integrin function, whereas K2 recruitment was unaffected. Our interdisciplinary approach also revealed that ILK does not directly bind {beta}1-integrin cytosolic domains in vitro and in cells. Instead, ILK was recruited to integrins exclusively through a K2-dependent mechanism, primarily via K2 bridging ILK and {beta}1 integrins. These findings define the hierarchical relationship between ILK and K2 in FAs and highlight the essential role of K2-mediated ILK recruitment for integrin adhesion and signaling. Significance StatementHow cells anchor to their environment is a fundamental question in biology. Integrins provide such a connection by bridging the extracellular matrix and the cytoskeleton. A central regulator of the integrin machine is Integrin-linked kinase (ILK). How ILK is recruited to {beta}1 integrins is hotly debated since its discovery more than 30 years ago. By integrating cell biology and biochemistry with super-resolution DNA-PAINT microscopy and a novel spatial analysis framework, we demonstrate that ILK does not bind directly to integrin cytoplasmic tails. Instead, we found that ILK is recruited by kindlin-2 (K2) to active, adhesion plaque-resident integrins. This work resolves a long-standing controversy in cell biology and establishes a versatile workflow for distinguishing direct from indirect protein-protein interactions in situ.

cell biology↗

bioRxiv2026

Preclinical safety assessment of Koomba kaat 1 and Biyabeda mokiny 1 phages for respiratory application against Staphylococcus aureus

Therapeutic bacteriophages are promising alternatives to antibiotics for treating antimicrobial-resistant bacterial infections. For pulmonary infections, direct respiratory delivery offers therapeutic advantages; however, preclinical evaluation of respiratory safety remains underdeveloped. Bacteriophages exhibit genomic and biological diversity, and safety cannot be assumed for individual candidates. Here, we evaluated the respiratory safety of lytic Staphylococcus aureus bacteriophages, Koomba kaat 1 and Biyabeda mokiny 1, using human and murine preclinical models. Differentiated primary airway epithelial cells derived from six healthy paediatric donors were exposed apically to purified phage (1 x 109 PFU/mL) for 24 hours. Barrier integrity, epithelial morphology, mucus production, cytotoxicity, and interleukin-8 release were assessed. Safety was further investigated in adult C57BL/6J mice receiving intranasal phage administration (1 x 109 PFU) twice daily (14 days). Clinical observations, body weight, organ pathology, blood biochemistry, bronchoalveolar lavage cellularity, protein concentration, and inflammatory mediators were evaluated. Neither phage altered epithelial morphology, barrier integrity, mucus production, cytotoxicity, nor inflammatory responses in differentiated cultures. Repeated intranasal administration was well tolerated in vivo, with no adverse clinical signs, weight loss, macroscopic pathology, or treatment-associated changes in pulmonary cellularity or tissue histopathology. Differences in blood biochemistry and inflammatory mediators were small and not accompanied by epithelial injury or pulmonary inflammation. Collectively, these findings demonstrate that Koomba kaat 1 and Biyabeda mokiny 1 exhibit favourable safety profiles following repeated airway administration. This study establishes a comprehensive framework for the preclinical respiratory safety assessment of bacteriophages and provides support for the clinical development of inhaled phage for S. aureus respiratory infections.

microbiology↗

bioRxiv2015

Avoiding accuracy-limiting pitfalls in the study of protein-ligand interactions with isothermal titration calorimetry

Isothermal titration calorimetry (ITC) can yield precise (3%) estimates of the thermodynamic parameters describing biomolecular association (affinity, enthalpy, and entropy), making it an indispensable tool for biochemistry and drug discovery. Surprisingly, interlaboratory comparisons suggest that errors of {bsim} 20% are common and widely underreported. Here, we show how to reduce precision- and accuracy-limiting errors while obtaining good estimates and minimizing material and time consumed by an experiment. We provide a simple spreadsheet that allows practitioners to identify precision-limiting operations during protocol design, track precision during the experiment, and propagate error to yield realistic final uncertainties.

Biochemistry↗

bioRxiv2016

An open library of human kinase domain constructs for automated bacterial expression

Kinases play a critical role in many cellular signaling pathways and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Since the FDA approval of imatinib in 2001, therapeutics targeting kinases now account for roughly 50% of current cancer drug discovery efforts. The ability to explore human kinase biochemistry, biophysics, and structural biology in the laboratory is essential to making rapid progress in understanding kinase regulation, designing selective inhibitors, and studying the emergence of drug resistance. While insect and mammalian expression systems are frequently used for the expression of human kinases, bacterial expression systems are superior in terms of simplicity and cost-effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression could produce high yields of Src and Abl kinase domains in bacterial expression systems, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 {micro}g/mL culture in a simple automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases, Lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identify kinase domain constructs previously expressed in bacteria likely to express well in a simple high-throughput protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate how the resulting expressing constructs can be used for the biophysical and biochemical study of clinical mutations by engineering a panel of 48 Src mutations and 46 Abl mutations via single-primer mutagenesis and screening the resulting library for expression yields. The wild-type kinase construct library is available publicly via Addgene, and should prove to be of high utility for experiments focused on drug discovery and the emergence of drug resistance.

Biochemistry↗

bioRxiv2016

Relative contributions of conformational selection and induced fit

A long standing debate in biochemistry is to determine whether the conformational changes observed during biomolecular interactions proceed through conformational selection (of preexisting isoforms) or induced fit (ligand-induced 3D reshaping). The latter mechanism had been invoked in certain circumstances, for example to explain the non-Michaelian activity of monomeric enzymes like glucokinase. But the relative importance of induced fit has been recently depreciated in favor of conformational selection, assumed to be always sufficient, predominant in general and in particular for glucokinase. The relative contributions of conformational selection and induced fit are reconsidered here in and out of equilibrium, in the light of earlier concepts such as the cyclic equilibrium rule and the turning wheel of Wyman, using single molecule state probability, one way fluxes and net fluxes. The conditions for a switch from conformational selection to induced fit at a given ligand concentration are explicitly determined. Out of equilibrium, the inspection of the enzyme states circuit shows that conformational selection alone would give a Michaelian reaction rate but not the established nonlinear behavior of glucokinase. Moreover, when induced fit and conformational selection coexist and allow kinetic cooperativity, the net flux emerging in the linkage cycle necessarily corresponds to the induced fit path.

Biochemistry↗

bioRxiv2016

p53 dynamically directs TFIID assembly on target gene promoters

The p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIIDs ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIIDs conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIIDs interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

biochemistry↗

bioRxiv2017

Toward D-Peptide Biosynthesis: Elongation Factor P Enables Ribosomal Incorporation Of Consecutive D-Amino Acids

To maintain stereospecific biochemistry in cells, living organisms have evolved mechanisms to exclude D-amino acids (DAA) in their protein synthesis machinery, which also limits our exploration of the realm of mirror-image molecules. Here, we show that high affinity between EF-Tu and aminoacyl-tRNA promotes D-amino acid incorporation. More strikingly, Elongation Factor P efficiently resolves peptidyl transferase stalling between two consecutive D-amino acids, and hence enables the translation of D-peptides.

biochemistry↗

bioRxiv2017

Multi-omic mitoprotease profiling defines a role for Oct1p in coenzyme Q production

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis--a pathway essential for mitochondrial respiration. Through Edman sequencing, and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N-terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Our custom data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.

biochemistry↗

bioRxiv2017

Evolution of an enzyme from a solute-binding protein

Much of the functional diversity observed in modern enzyme superfamilies originates from molecular tinkering with existing enzymes1. New enzymes frequently evolve from enzymes with latent, promiscuous activities2, and often inherit key features of the ancestral enzyme, retaining conserved catalytic groups and stabilizing analogous intermediates or transition states3. While experimental evolutionary biochemistry has yielded considerable insight into the evolution of new enzymes from existing enzymes4, the emergence of catalytic activity de novo remains poorly understood. Although certain enzymes are thought to have evolved from non-catalytic proteins5-7, the mechanisms underlying these complete evolutionary transitions have not been described. Here we show how the enzyme cyclohexadienyl dehydratase (CDT) evolved from a cationic amino acid-binding protein belonging to the solute-binding protein (SBP) superfamily. Analysis of the evolutionary trajectory between reconstructed ancestors and extant proteins showed that the emergence and optimization of catalytic activity involved several distinct processes. The emergence of CDT activity was potentiated by the incorporation of a desolvated general acid into the ancestral binding site, which provided an intrinsically reactive catalytic motif, and reshaping of the ancestral binding site, which facilitated enzyme-substrate complementarity. Catalytic activity was subsequently gained via the introduction of hydrogen-bonding networks that positioned the catalytic residue precisely and contributed to transition state stabilization. Finally, catalytic activity was enhanced by remote substitutions that refined the active site structure and reduced sampling of non-catalytic states. Our work shows that the evolutionary processes that underlie the emergence of enzymes by natural selection in the wild are mirrored by recent examples of computational design and directed evolution of enzymes in the laboratory.

biochemistry↗

bioRxiv2017

DddY is a bacterial dimethylsulfoniopropionate lyase representing a new cupin enzyme superfamily with unknown primary function

Dimethylsulfide (DMS) is released at rates of >107 tons annually and plays a key role in the oceanic sulfur cycle and ecology. Marine bacteria, algae, and possibly other organisms, release DMS via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known belonging to different superfamilies and exhibiting highly variable levels of DMSP lyase activity. DddY shows the highest activity among all reported bacterial lyases yet is poorly characterized. Here, we describe the characterization of recombinant DddY is from different marine bacteria. We found that DddY activity demands a transition metal ion cofactor. DddY also shares two sequence motifs with other bacterial lyases assigned as cupin-like enzymes, DddQ, DddL, DddK, and DddW. These cupin motif residues are essential for DddY activity, as for the other cupin DMSP lyases, and all these enzymes are characterized by a common metal-chelator inhibitor (TPEN). Analysis of all sequences carrying these cupin motifs defined a superfamily: Cupin-DLL (DMSP lyases and lyase-like). The DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily independently along multiple lineages. However, the specific activity levels, genomic context analysis, and systematic profiling of substrate selectivity as described in the accompanying paper, indicate that for only some of these families, most distinctly DddY and DddL, DMSP lyase is the primary, native activity. In other families, foremost DddQ, DMSP lyase seems to be merely a promiscuous activity. The native function of DddQ, and of nearly all members of this newly identified Cupin-DLL superfamily, remains unknown.\n\nAbbreviations\n\nFundingFinancial support by the Estate of Mark Scher, and the Sasson & Marjorie Peress Philanthropic Fund, are gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

biochemistry↗

bioRxiv2017

Native or promiscuous? Analyzing putative dimethylsulfoniopropionate lyases using a substrate proofing approach

Enzyme promiscuity is widely spread. Foremost, within superfamilies, the native function of one enzyme is typically observed as promiscuous activity in related enzymes. The native function usually exhibits high catalytic efficiency while promiscuous activities are weak, but this is not always the case. Thus, for certain enzymes it remains questionable whether their currently known activity is native or promiscuous. Dimethylsulfon-iopropionate (DMSP) is an abundant marine metabolite cleaved via {beta}-elimination to release dimethylsulfide (DMS). Eight different gene families have been identified as putative DMSP lyases, 5 of them belonging to the same superfamily (cupin-DLL; see the accompanying paper). Some of these enzymes exhibit very low activity, but this can be due to suboptimal folding or reaction conditions. We developed a substrate profiling approach with the aim of distinguishing native DMSP lyases from enzymes that promiscuously act as DMSP lyases. In a native DMSP lyase, relatively small changes in the structure of DMSP should induce significant activity drops. We thus profiled substrate selectivity by systematically modifying DMSP while retaining reactivity. Three enzymes that exhibit the highest activity with DMSP also exhibited high sensitivity to perturbation of DMSPs structure (Alma, DddY, and DddL). The two enzymes with the weakest DMSP lyase activity also showed the highest crossreactivity (DddQ, DddP). Combined with other indications, it appears that the DMSP lyase activity of DddQ and DddP is promiscuous although their native function remains unknown. Systematic substrate profiling could help identify and assign potential DMSP lyases, and possibly applied to other enzymes.\n\nAbbreviationsDMSP, dimethylsulfoniopropionate; DMS, dimethylsulfide; cupin-DLL, cupin DMSP lyase and lyase-like.\n\nFundingFinancial support by the Estate of Mark Scher, and the Sasson & Marjorie Peress Philanthropic Fund, are gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

biochemistry↗

bioRxiv2017

Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, which has historically been the thought for the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that medium-sized tightly folded proteins such as the 22 kDa dihydrofolate reductase (DHFR) can be tolerated by TOC and TIC. Chimeric DHFR fused with RuBisCO small subunit transit peptide (tp22DHFR) was found to be imported into chloroplasts in complex with its stabilizing ligand, methotrexate (MTX), in a folded conformation. Following import, both mature tp22DHFR and MTX were found in the chloroplast stroma. A subsaturating concentration of MTX was used to exclude the possibility that MTX was stripped off tp22DHFR, independently imported into the chloroplasts, and reassociated with imported tp22DHFR. Independent MTX import was further excluded by use of fluorescein conjugated MTX (FMTX), which has very slow membrane transport rates relative to unconjugated MTX. The TOC/TIC pore size was determined by probing the translocons with particles of fixed diameter and found to be greater than 25.6 [A], large enough to support folded DHFR import. The pore size is also larger than those of the mitochondrial protein translocons that have a requirement for protein unfolding.\n\nSIGNIFICANCEThe chloroplast TOC and TIC translocons are responsible for the import of up to 95% of all chloroplast proteins and are therefore essential for plastid biogenesis and photosynthesis. However, the mechanisms of protein import into chloroplasts are not well understood. The TOC/TIC translocons have long been suggested to have a strong unfoldase activity relative to other comparable protein translocons. Here, we present data suggesting that this is not true, and that instead, they possess a relatively large pore size. This identifies TOC and TIC as rather unique protein translocons capable of transporting folded proteins across a double membrane barrier, which has important implications in the mechanisms of TOC/TIC function and biogenesis of photosynthetic proteins.\n\nClassification - Biochemistry

biochemistry↗

bioRxiv2017

RNA binding and HEPN nuclease activation are decoupled in CRISPR-Cas13a

CRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13as target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central binding seed where perfect base pairing is absolutely required for target binding, and a separate nuclease switch where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13as RNA binding and cleavage behavior for RNA-targeting tool development.

biochemistry↗

bioRxiv2017

The importance of the photosynthetic Gibbs effect in the elucidation of the Calvin-Benson-Bassham Cycle

The photosynthetic carbon reduction cycle, or Calvin-Benson-Bassham Cycle, is now contained in every standard biochemistry textbook. Although the cycle was already proposed in 1954, it is still subject of intense research, and even the structure of the cycle, i.e. the exact series of reactions, is still under debate. The controversy about the cycles structure was fuelled by the findings of Gibbs and Kandler in 1956 and 1957, when they observed that radioactive 14CO2 was dynamically incorporated in hexoses in a very atypical and asymmetrical way, a phenomenon later termed the photosynthetic Gibbs effect. Now, it is widely accepted that the photosynthetic Gibbs effect is not in contradiction to the reaction scheme proposed by Calvin, Benson and Bassham, but the arguments given have been largely qualitative and hand-waving. To fully appreciate the controversy and to understand the difficulties in interpreting the Gibbs effect, it is illustrative to illuminate the history of the discovery of the Calvin-Benson-Bassham Cycle. We here give an account of central scientific advances and discoveries, which were essential prerequisites for the elucidation of the cycle. Placing the historic discoveries in the context of the modern textbook pathway scheme illustrates the complexity of the cycle and demonstrates why especially dynamic labelling experiments are far from easy to interpret. We conclude by arguing that only mathematical models based on a sound theory are capable of resolving conflicting interpretations and providing a consistent quantitative explanation.

biochemistry↗

bioRxiv2017

Error-prone bypass of DNA lesions during lagging strand replication is a common source of germline and cancer mutations

Spontaneously occurring mutations are of great relevance in diverse fields including biochemistry, oncology, evolutionary biology, and human genetics. Studies in experimental systems have identified a multitude of mutational mechanisms including DNA replication infidelity as well as many forms of DNA damage followed by inefficient repair or replicative bypass. However, the relative contributions of these mechanisms to human germline mutations remain completely unknown. Here, based on the mutational asymmetry with respect to the direction of replication and transcription, we suggest that error-prone damage bypass on the lagging strand plays a major role in human mutagenesis. Asymmetry with respect to transcription is believed to be mediated by the action of transcription-coupled DNA repair (TC-NER). TC-NER selectively repairs DNA lesions on the transcribed strand; as a result, lesions on the non-transcribed strand are preferentially converted into mutations. In human polymorphism we detect a striking similarity between transcriptional asymmetry and asymmetry with respect to replication fork direction. This parallels the observation that damage-induced mutations in human cancers accumulate asymmetrically with respect to the direction of replication, suggesting that DNA lesions are asymmetrically resolved during replication. Re-analysis of XR-seq data, Damage-seq data and cancers with defective NER corroborate the preferential error-prone bypass of DNA lesions on the lagging strand. We experimentally demonstrate that replication delay greatly attenuates the mutagenic effect of UV-irradiation, in line with the key role of replication in conversion of DNA damage to mutations. We conservatively estimate that at least 10% of human germline mutations arise due to DNA damage rather than replication infidelity. The number of these damage-induced mutations is expected to scale with the number of replications and, consequently, paternal age.

biochemistry↗

bioRxiv2019

Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

biochemistry↗