Biology subjects

Tainer, J. A.

Publications and source records attributed to Tainer, J. A..

11 recordsLinked to original sources

bioRxiv2026

Orchestrated metal ion repositioning defines the dynamic catalytic strategy of the essential DNA repair nuclease APE1

Human AP-endonuclease 1 (APE1) is a vital enzyme in the base excision repair pathway that protects genome stability by eliminating ubiquitous abasic DNA lesions. Despite its importance and therapeutic potential, how APE1 achieves high specificity and single-metalion catalytic efficiency remains unclear. Here, we present a high-resolution structure of the APE1-DNA Michaelis complex coordinated with physiological cofactor Mg2+. Integrating this snapshot with ab initio molecular dynamics and metadynamics simulations reveals a novel "moving metal ion" mechanism in which Mg2+ undergoes orchestrated repositioning to trigger a concerted catalytic reaction, bypassing the formation of an associative pentavalent intermediate. This distinct catalytic strategy, driven by concerted active-site reorganization, enables APE1 to efficiently process damaged DNA using only one metal ion cofactor. A previously unrecognized hydrogen-bonding network couples catalytic water activation to the metal ion movement - two events that strikingly occur on opposite sides of the active site. These findings provide a blueprint for how enzymes synchronize distal active site rearrangements with transition state formation. Our results further suggest that effective AI-targeted inhibitor design should develop capacities to predict mechanistically critical non-canonical rotamers and transient hydrogen-bonding networks. Our combined findings offer a foundation for the design of inhibitors targeting APE1 overexpression in cancer.

biochemistry↗

bioRxiv2025

Human CDK5-cyclin B1 structures uncover a conserved mitotic CDK activation mechanism

Cyclin-dependent kinase 5 (CDK5), long considered atypical and activated by non-cyclin cofactors in neurons, also functions in mitosis. To resolve its activation mechanism during mitosis, we determined the first high-resolution crystal structures of CDK5-cyclin B1 in apo and nucleotide-bound states. Contrary to AlphaFold predictions, we find CDK5 structures unexpectedly mirrors CDK1-cyclin B1 assembly and activation, establishing CDK5 as a bona fide mitotic kinase acting in parallel with CDK1.

biochemistry↗

bioRxiv2025

Huntingtin preserves mitochondrial genome integrity in neurons, which is impaired in Huntingtons disease

Huntingtin (HTT) function is enigmatic, as the native protein plays critical roles in neuronal health, while mutant HTT (mHTT), carrying an expanded polyglutamine stretch, triggers neurotoxicity and contributes to the pathogenesis of Huntingtons disease (HD). We recently found that HTT is part of a nuclear transcription-coupled DNA repair (TCR) complex with DNA repair enzymes including polynucleotide-kinase-3-phosphatase (PNKP). This complex resolves DNA lesions during transcription to maintain genome integrity, while in HD, mHTT impairs the activity of this complex, resulting in accumulation of DNA lesions. Using molecular, cellular biology and computational methods, we find that HTT has a role in assembling a functional DNA repair complex in mitochondria. Together with mitochondrial RNA polymerase and transcription factors, HTT resolves mitochondrial DNA lesions to preserve mitochondrial genome integrity and function. Pathogenic mHTT impairs this activity, resulting in persistent DNA lesions and reduced mitochondrial function in HD. Importantly, restoring activity of this complex in a Drosophila HD model through ectopic HTT or PNKP expression significantly improves mitochondrial genome integrity and ameliorates motor deficits. HIGHLIGHTSO_LIHTT organizes a functional, multifactorial mitochondrial DNA repair complex C_LIO_LIMutant HTT impairs the mitochondrial DNA repair complex causing DNA damage accumulation C_LIO_LIHTT-associated repair complex resolves mitochondrial DNA lesions and DNA integrity C_LIO_LIRestoring repair activity in HD flies rescues mitochondrial DNA integrity and motor defects C_LI

neuroscience↗

bioRxiv2024

Information Theory Optimization of Signals from Small Angle Scattering Measurements

Solution-state small angle scattering (SAS) using X-rays (SAXS) or neutrons (SANS), informs on the conformational states and assemblies of biological macromolecules (bioSAS) outside of cryo- and solid-state conditions. BioSAS measurements are resolution-limited and through an inverse Fourier transform, the measured SAS intensities directly relate to the physical space occupied by the particles via the P(r)-distribution. Yet, this inverse transform of SAS data is canonicaly cast as an ill-posed, ill-conditioned problem requiring an indirect approach. Here, we show that with modern instrumentation and the application of information theory, the inverse transform of SAXS intensity data can be cast as a well-conditioned problem and that the ill-conditioning of the inverse problem is directly related to the Shannon number. By exploiting the oversampling enabled by modern detectors, a direct inverse Fourier transform of the SAXS data is possible provided the recovered information does not exceed the Shannon limit. The limit corresponds to the maximum number of significant singular values that can be recovered in a SAXS experiment suggesting the relationship between the Shannon limit and significant singular values is a fundamental property of band-limited inverse integral transform problems. An intrinsic challenge to the type of problem found in SAS, is identifying the best model while avoiding over-fitting. We propose a hybrid scoring function employing an information theory framework that assesses both the quality of the model-data fit as well as the quality of the recovered P(r)-distribution. The hybrid score utilizes the Akaike Information Criteria and Durbin-Watson (DW) statistic that considers model complexity, i.e., degrees-of-freedom and randomness of the model-data residuals. Importantly, the described tests and findings extend the boundaries for bioSAXS by completing the information theory formalism initiated by Peter B. Moore to enable a quantitative measure of resolution in SAS, robustly determine maximum dimension, and more precisely define the best model to appropriately represent the observed scattering data. SIGNIFICANCEThe inverse transform problem in small angle scattering (SAS) of dispersed particles in solution is canonically considered an ill-posed, ill-conditioned problem. We apply information theory to show that the so-called Shannon number, Ns, of band-width limited signals defines the boundary at which a problem transitions from well-conditioned to ill-conditioned. In this framework, Ns represents the maximum number of orthogonal elements that are available to the SAS measurement and defines a fundamental relationship that determines the effective resolution of the recovered P(r)-distribution. We find that the inverse problem can be solved directly using a Shannon-limited approach. The Shannon-limit establishes model complexity and when incorporated into the Akaike Information Criteria aids in selecting the most likely P(r)-distribution.

biophysics↗

bioRxiv2024

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2is FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

molecular biology↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗

bioRxiv2023

The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the antiviral innate immune response

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non- structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5- triphosphorylated double-stranded RNA (5ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5ppp-dsRNA, and thus suppresses activation of RIG-Is ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

biochemistry↗

bioRxiv2023

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

We developed a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-validation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

biochemistry↗

bioRxiv2023

Dynamics of the DYNLL1/MRE11 complex regulates DNA end resection and recruitment of the Shieldin complex to DSBs

Extent and efficacy of DNA end resection at DNA double strand break (DSB)s determines the choice of repair pathway. Here we describe how the 53BP1 associated protein DYNLL1 works in tandem with Shieldin and the CST complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1 where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1 predominantly in the G1 cells. Shieldin localization to DSBs is dependent on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be re-sensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

molecular biology↗

bioRxiv2022

Two Interaction Surfaces between XPA and RPA Organize the Preincision Complex in Nucleotide Excision Repair

The XPA and RPA proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair pathway. We have previously characterized the two interaction surfaces between XPA and RPA, with the RPA32 and RPA70AB subunits. Here we show that the mutations in the two individual interaction surfaces reduce NER activity in biochemical and cellular systems, and that combining mutations in two domains leads to an additive inhibition of NER, suggesting that they fulfill distinct roles. Our data suggest that the interaction between XPA and RPA32 is important for the initial association of XPA with NER complexes, while the interaction between XPA and RPA70 is needed for structural organization of the complex to license the dual incision reaction. SAXS analysis of complexes of XPA and RPA bound to ss/dsDNA junction substrates reveals the architecture of XPA and RPA in the preincision complex and shows that the two interaction domains between RPA and XPA are located at opposite sides of the two molecules. We propose a structure for the overall NER preincision complex that shows that the two strands of the NER bubble assume a U-shape with the two ss/dsDNA junctions localized in close proximity, with the interaction between XPA and RPA70 as one of the key organizing elements.

biochemistry↗

bioRxiv2020

Polymerase Theta Inhibition Kills Homologous Recombination Deficient Tumors

PARP inhibitors (PARPi) have become a new line of therapy for Homologous Recombination (HR)-deficient cancers. However, resistance to PARPi has emerged as a major clinical problem. DNA polymerase theta (POL{theta}) is synthetic lethal with HR and a druggable target in HR-deficient cancers. Here, we identified the antibiotic Novobiocin (NVB) as a specific POL{theta} inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POL{theta} ATPase domain, inhibits its ATPase activity, and phenocopies POL{theta} depletion. BRCA-deficient tumor cells and those with acquired PARPi resistance are sensitive to NVB in vitro and in vivo. Increased POL{theta} expression levels predict NVB sensitivity. The mechanism of NVB-mediated cell death in PARPi resistant cells is the accumulation of toxic RAD51 foci, which also provides a pharmacodynamic biomarker for NVB response. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. One Sentence SummaryWe identified Novobiocin as a specific POL{theta} inhibitor that selectively kills naive and PARPi resistance HR-deficient tumors in vitro and in vivo.

cancer biology↗