Biology subjects

Weaver, T. M.

Publications and source records attributed to Weaver, T. M..

14 recordsLinked to original sources

bioRxiv2026

Structural basis of end processing in the nucleosome by polynucleotide kinase phosphatase

Genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Chromatinized DNA is under constant assault from endogenous and exogenous sources of damage, which must be effectively repaired to preserve genome stability. Single-strand breaks (SSBs) with chemically heterogeneous DNA ends are one of the most prevalent forms of genomic DNA damage. These SSBs must be enzymatically processed prior to downstream gap-filling DNA synthesis and/or nick ligation during single-strand break repair (SSBR). Polynucleotide kinase phosphatase (PNKP) is a multifunctional end-processing enzyme that possesses two catalytic activities important for converting non-ligatable SSBs into ligatable SSBs. To date, a mechanistic description for how PNKP processes non-ligatable SSBs in the context of chromatin to initiate SSBR remains undefined. Here, we utilize a combination of biochemical assays and cryogenic electron microscopy (cryo-EM) to define the structural basis of end processing in the nucleosome by PNKP. Cryo-EM structures of PNKP engaged with non-ligatable SSBs at three unique positions within the nucleosome reveal that PNKP locally deforms nucleosomal DNA to reposition the SSBs into the kinase and phosphatase active sites, providing a structural basis for the efficient processing of SSBs throughout the nucleosome. Additional cryo-EM structures reveal the PNKP FHA domain also engages the nucleosome acidic patch during non-ligatable SSB recognition, which accelerates the processing of non-ligatable SSBs in the nucleosome. Together, these findings provide important mechanistic insight into the initial end processing step of chromatin-based SSBR.

biochemistry↗

bioRxiv2026

Molecular basis of nick ligation in the nucleosome by DNA Ligase IIIα

Eukaryotic genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Within this chromatin context, genomic DNA is constantly exposed to endogenous and exogenous stress that result in the formation of DNA damage, which must be effectively repaired to maintain genome stability. Single-strand breaks (SSBs) are among the most prevalent forms of DNA damage that arise via the oxidation-induced disintegration of the sugar-phosphate backbone or as repair intermediates during base excision repair. DNA ligase III (LigIII) is one of the primary enzymes responsible for repairing SSBs containing an intact 5'-phosphate and 3'-OH (nick) during the terminal step of single-strand break repair (SSBR) and base excision repair (BER) pathways. To date, a complete mechanistic description for how LigIII processes nicks within chromatin remains elusive. Here, we use a combination of biochemical assays, molecular dynamics simulations, and cryogenic electron microscopy (cryo-EM) to define the molecular basis of nick ligation in the nucleosome by LigIII. Quantitative enzyme kinetics reveal that the LigIII ligation rate is highly dependent on the translational position of the nick in the nucleosome, where nicks near the nucleosome entry/exit site are ligated with moderate efficiency and nicks near the nucleosome dyad are refractory to ligation. Cryo-EM structures of LigIII bound to nicks at four unique translational positions in the nucleosome reveal the structural basis for this position-dependent catalytic activity, identifying that local steric constraints imposed by the histone octamer prevent LigIII from readily adopting a ligation-competent conformation. Further biochemical and structural analysis demonstrates that the scaffolding protein XRCC1, which forms a heterodimer with LigIII, does not substantially alter the ability of LigIII to bind or ligate nicks in the nucleosome. Together, this work provides foundational insight into the processing of nicks in the nucleosome during the terminal step of SSBR/BER.

biochemistry↗

bioRxiv2025

Fork Reversal Safeguards Epigenetic Inheritance during DNA Replication Under Stress

During DNA replication, epigenetic information carried by histone modifications is faithfully propagated and re-established on sister chromatids, ensuring cell identity. Chromatin reassembly is tightly coupled to DNA replication, therefore whether and how perturbations to DNA replication affects the fidelity of epigenetic inheritance remain poorly understood. In this study, we reveal a critical role for replication fork reversal in maintaining the transmission of epigenetic information under replication stress. We identify that cells defective in fork reversal exhibit reduced nucleosome density at replication forks, accompanied by the loss of parental histones during their transfer onto nascent DNA. Mechanistically, we demonstrate that PrimPol activation leads to single-stranded DNA gaps in fork reversal deficient cells, and that subsequent PARylation (poly ADP-ribosylation) and DNA-protein crosslinking on these gaps evicts nucleosomes. Our findings demonstrate that replication fork reversal, a widespread physiological process, is not only essential for preserving genome integrity but also for safeguarding epigenetic stability.

biochemistry↗

bioRxiv2025

Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.

biophysics↗

bioRxiv2024

Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle

Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.

biochemistry↗

bioRxiv2024

Structural basis of gap-filling DNA synthesis in the nucleosome by DNA Polymerase β

Single-strand breaks (SSBs) are one of the most prevalent forms of DNA damage found in the chromatinized genome and are repaired by direct single-strand break repair (SSBR) or base excision repair (BER). DNA polymerase beta (Pol {beta}) is the primary enzyme responsible for processing the 1-nt gap intermediate in chromatin during SSBR and BER. However, the mechanism used by Pol {beta} to process a 1-nt gap in the context of the nucleosome and chromatin remains poorly understood. Here, we use biochemical assays and cryogenic electron microscopy (cryo-EM) to determine the kinetic and structural basis of gap-filling DNA synthesis in the nucleosome by Pol {beta}. Kinetic analysis identified that gap-filling DNA synthesis in the nucleosome by Pol {beta} is position-dependent, where solvent exposed 1-nt gaps are processed more efficiently than histone-occluded 1-nt gaps. A series of cryo-EM structures of Pol {beta} bound to a solvent-exposed 1-nt gap in the nucleosome reveal a global DNA sculpting mechanism for 1-nt gap recognition, which is mediated by sequential engagement of the Pol {beta} lyase domain and polymerase domain. Finally, cryo-EM structures of Pol {beta} bound to 1-nt gaps at two additional positions in the nucleosomal DNA define the structural basis for position-dependent nucleotide insertion in the nucleosome. This work establishes the mechanism used by Pol {beta} for processing 1-nt gaps in the nucleosome during SSBR and BER, providing fundamental insight into DNA repair in chromatin.

biochemistry↗

bioRxiv2024

Spatial Deconvolution of Cell Types and Cell States at Scale Utilizing TACIT

Identifying cell types and states remains a time-consuming and error-prone challenge for spatial biology. While deep learning is increasingly used, it is difficult to generalize due to variability at the level of cells, neighborhoods, and niches in health and disease. To address this, we developed TACIT, an unsupervised algorithm for cell annotation using predefined signatures that operates without training data, using unbiased thresholding to distinguish positive cells from background, focusing on relevant markers to identify ambiguous cells in multiomic assays. Using five datasets (5,000,000-cells; 51-cell types) from three niches (brain, intestine, gland), TACIT outperformed existing unsupervised methods in accuracy and scalability. Integration of TACIT-identified cell with a novel Shiny app revealed new phenotypes in two inflammatory gland diseases. Finally, using combined spatial transcriptomics and proteomics, we discover under- and overrepresented immune cell types and states in regions of interest, suggesting multimodality is essential for translating spatial biology to clinical applications.

bioinformatics↗

bioRxiv2024

Hierarchical determinants of the oxidation-induced mutational landscape in human cells.

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion, which causes G>T substitutions that compose COSMIC single base substitution signature 18 (SBS18) in human cancers. Determinants of local and regional differences in 8-oxoG-induced mutability are currently unknown. To uncover factors influencing the topology of 8-oxoG-induced mutations, we assessed spontaneous and KBrO3-induced 8-oxoG mutagenesis in human cell lines. KBrO3 exposure produced a SBS18-like substitution spectrum and a distinct never-before reported INDEL signature that we also observed in human cancers. KBrO3-induced 8-oxoG lesions occurred with similar sequence preference as KBrO3-induced substitutions, indicating that the reactivity of specific reactive oxygen species (ROS) dictates the trinucleotide motif specificity for 8-oxoG-induced mutagenesis. While 8-oxoG lesions occurred relatively uniformly across chromatin states and nucleosomes, 8-oxoG-induced mutations occurred more frequently in more compact regions of the genome, within nucleosomal DNA, and at inward facing guanines within strongly positioned nucleosomes. Cryo-EM structures of OGG1 bound to nucleosomes indicate that these effects originate from OGG1s ability to flip outward positioned 8-oxoG lesions into the catalytic pocket with only minor alterations to nucleosome structure, while inward facing lesions occluded by the histone octamer are unrecognized. Mutation spectra from cells with DNA repair deficiencies revealed a hierarchical DNA repair network limiting 8-oxoG mutagenesis in human cells, where OGG1- and MUTY-mediated BER is supplemented by replication-associated factors participating in tolerance of 8-oxoG or derived repair intermediates (i.e. Pol {eta} and HMCES). Surprisingly, analysis of transcriptional asymmetry of KBrO3-induced mutations demonstrated transcription-coupled repair of 8-oxoG in Pol {eta}-deficient cells. Thus, radical chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.

cancer biology↗

bioRxiv2023

Single-molecule analysis of purified proteins and nuclear extracts: insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

biochemistry↗

bioRxiv2023

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.

biophysics↗

bioRxiv2022

Structural basis for APE1 processing DNA damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determined that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site identified that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identified contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

biochemistry↗

bioRxiv2021

Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1.

biochemistry↗

bioRxiv2021

REPROGRAMMING CBX8-PRC1 FUNCTION WITH A POSITIVE ALLOSTERIC MODULATOR

ABSTRACTCanonical targeting of Polycomb Repressive Complex 1 (PRC1) to repress developmental genes is mediated by cell type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7 and 8). Based on their central role in silencing and their misregulation associated with human disease including cancer, CBX proteins are attractive targets for small molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids and participation in variant PRC1. We show that treatment with UNC7040 leads to efficient PRC1 chromatin eviction, loss of silencing and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only revealed the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of polycomb regulation by non-histone lysine methylated interaction partners.

synthetic biology↗

bioRxiv2020

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites as well as minor-groove and exocyclic guanine adducts. It does this by using a unique protein-template mechanism in which the template base is flipped out of the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain. To observe Rev1 catalysis at the atomic level, we employed time-lapse X-ray crystallography. We found that Rev1 flips out the template base prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, and this is not coupled to large structural changes in the protein like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono-phosphates, which drives the reaction in the forward direction. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These post-catalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

biochemistry↗