Biology subjects

Whitaker, A. M.

Publications and source records attributed to Whitaker, A. M..

5 recordsLinked to original sources

bioRxiv2026

Cohesin residence time gates 3D genome response to histone hyperacetylation

Cohesin-mediated loop extrusion and chromatin state-dependent compartmentalization are major drivers of three-dimensional (3D) genome organization. Although epigenomic perturbations are widely assumed to reshape chromatin architecture, the mechanisms that determine how changes in chromatin state are translated into structural reorganization remain poorly understood. Here, we identify cohesin residence time as a key regulator of the genomes architectural response to histone hyperacetylation induced by histone deacetylase inhibition (HDACi). Acute depletion of RAD21 or CTCF weakens chromatin loops but preserves HDACi-induced changes in compartmentalization, contact-scaling behavior, and loop density. In contrast, perturbation of cohesin loading or release produces opposing effects: NIPBL depletion sensitizes and amplifies architectural responses to HDACi, whereas WAPL loss renders the genome largely refractory to HDACi-induced remodeling, suppressing changes in compartments and loop density while stabilizing CTCF-anchored loops. These distinct architectural outcomes occur despite comparable levels of HDACi-induced histone hyperacetylation across genotypes, indicating that differential epigenomic input is not responsible for the observed effects. Together, our findings demonstrate that dynamic cohesin turnover, rather than cohesin chromatin association alone, governs whether epigenomic perturbations are converted into higher-order genome reorganization. These results establish cohesin residence time as a molecular gate linking chromatin state to 3D genome architecture and reveal a previously unrecognized principle underlying chromatin architecture plasticity.

genomics↗

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↗

bioRxiv2023

The VEGF G-quadruplex forming promoter is repaired via long-patch BER

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased [~]2-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol {beta} performs multiple insertions on G4 substates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol {beta} products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol {beta} prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

biochemistry↗

bioRxiv2021

Processing of oxidatively damaged DNA dirty ends by APE1

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3 to 5 exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3-end of a DNA strand during replication or repair. Importantly, 3-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

biochemistry↗

bioRxiv2021

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a 3-color prismTIRF microscope which can be utilized for the study macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology to a broader spectrum of scientific questions.

biochemistry↗