Biology subjects

Sensoy, O.

Publications and source records attributed to Sensoy, O..

6 recordsLinked to original sources

bioRxiv2026

Phosphorylation-dependent Remodeling of the CLOCK/BMAL1/nucleosome Complex

Phosphorylation of the CLOCK/BMAL1 complex is a reversible post-translational modification that plays a central role in regulating circadian oscillations; however, its underlying mechanistic basis remains poorly understood. Although biochemical studies have shown that phosphorylation modulates CLOCK/BMAL1 binding to DNA, yet it remains unclear whether these effects are confined to local perturbation or also propagate through allosteric effects. Moreover, the influence of phosphorylation on histone dynamics and transcription factor-nucleosome interactions has not been systematically investigated. Here, we address these questions using atomistic trajectories obtained through backmapping of coarse-grained molecular dynamics simulations based on the recently resolved cryo-EM structure of the CLOCK/BMAL1 and nucleosome complex. We investigated three experimentally identified phosphorylation states: CLOCK bHLHS38/42, BMAL1 bHLHS78, and simultaneous phosphorylation of both proteins. Our results demonstrate that phosphorylation regulates CLOCK and BMAL1 asymmetrically. Whereas phosphorylation weakens the interaction of the modified bHLH domain with the E-box, BMAL1 phosphorylation simultaneously enhances DNA engagement by the CLOCK bHLH domain, an effect that persists in the doubly phosphorylated complex and identifies BMAL1 phosphorylation as the dominant regulatory event. Steered pulling simulations further demonstrate that phosphorylation equalizes the mechanical stability of CLOCK and BMAL1 interactions with DNA. Beyond modulating DNA binding, phosphorylation remodels protein-histone interactions by altering contacts between the CLOCK PASB domain and histone H3 and between the BMAL1 PASA domain and the H2A/H2B acidic patch, while simultaneously rewiring residue-correlation and allosteric communication networks throughout the heterodimer. Importantly, phosphorylation increases the separation between the CLOCK HI loop and the H3 1-L1 elbow, supporting a structural model in which phosphorylation acts as a priming event that provides a more permissive environment for CRY1 recruitment to the chromatin-bound CLOCK-BMAL1 complex, thereby facilitating transcriptional repression. Collectively, our findings reveal that phosphorylation regulates the CLOCK-BMAL1 complex through coordinated remodeling of DNA binding, nucleosome interactions, and long-range allosteric communication, providing a mechanistic framework for circadian transcriptional repression and a foundation for the rational design of therapeutics targeting the molecular circadian clock.

biophysics↗

bioRxiv2026

Polynucleotide Phosphorylase Stops Replication Restart by Degrading the RNA within R-loops during Replication-Transcription Conflicts

DNA replication and transcription machineries function simultaneously, on the same template, leading to conflicts between the two. During conflicts, the nascent mRNAs can hybridize to their template strands, generating RNA:DNA hybrids, which reside within nucleic acid structures known as R-loops. Accumulation of R-loops can lead to severe replication fork stalling, eventually leading to cell death. In bacteria, there are well-known replication restart mechanisms that should rescue these stalled replication forks, and yet, during conflicts, this process is inhibited. Additionally, in vitro work has shown that RNA:DNA hybrids are substrates for replication restart. Here, we discovered that the highly conserved exonuclease Polynucleotide Phosphorylase (PNPase in Bacillus subtilis) prevents replication restart from RNA:DNA hybrids, specifically at regions of severe conflicts where R-loops accumulate. Our in vitro data show that PNPase binds RNA:DNA hybrids and digests the RNA in these structures. Consistently, we found that in vivo, PNPase binds to conflict regions and reduces R-loop levels. To our knowledge, the only other class of enzymes known to digest the RNA from hybrids are RNases H. Our findings identify PNPase as a new enzyme that can perform a similar function. Importantly, our data show that PNPase inhibits replication restart from R-loops and its absence allows for replication restart from conflict regions. We also observed that PNPase activity reduces mutations, suggesting that replication restart from RNA:DNA hybrids is highly mutagenic. We propose that PNPase acts as a safeguard against mutagenic replication restart from RNA:DNA hybrids within R-loops by digesting the RNA which could re-initiate replication.

genetics↗

bioRxiv2026

A universal regulatory mechanism for prevention of replication restart from RNA:DNA hybrids

RNA:DNA hybrids form across all genomes. In vitro studies showed that these structures can be used to restart stalled replication forks, especially upon replication-transcription conflicts, however, this has not been tested in vivo. Here, we identify a mechanism that prevents replication restart from hybrids within cells. We identified the tRNA ligase, AsnRS, as a regulator of replication restart from hybrids at conflict regions in Bacillus subtilis. We determined that the DNA translocase Mfd and DNA polymerase PolA play key roles in this pathway. We unraveled a mechanism whereby Mfd removes RNA Polymerases, exposing 3 ends of the RNAs within hybrids. These 3 ends are then capped by AsnRS, preventing restart by PolA. Remarkably, the mammalian homologs of AsnRS, NARS1 and NARS2, fully complement the AsnRS phenotypes in bacteria. We propose that this is a universally conserved mechanism that prevents untimely replication initiation outside of origins from bacteria to humans.

genetics↗

bioRxiv2024

A small molecule enhances arrestin-3 binding to the β2-adrenergic receptor

G protein-coupled receptor (GPCR) signaling is terminated by arrestin binding to a phosphorylated receptor. Binding propensity has been shown to be modulated by stabilizing the pre-activated state of arrestin through point mutations or C-tail truncation. Here, we hypothesize that pre-activated rotated states can be stabilized by small molecules, and this can promote binding to phosphorylation-deficient receptors, which underly a variety of human disorders. We performed virtual screening on druggable pockets identified on pre-activated conformations in Molecular Dynamics trajectories of arrestin-3, and found a compound targeting an activation switch, the back loop at the inter-domain interface. According to our model, consistent with available biochemical and structural data, the compound destabilized the ionic lock between the finger and the back loop, and enabled transition of the gate loop towards the pre-activated state, which stabilizes pre-activated inter-domain rotation. The predicted binding pocket is consistent with saturation-transfer difference NMR data indicating close contact between the piperazine moiety of the compound and C/finger loops. The compound increases in-cell arrestin-3 binding to phosphorylation-deficient and wild-type {beta}2-adrenergic receptor, but not to muscarinic M2 receptor, as verified by FRET and NanoBiT. This study demonstrates that the back loop can be targeted to modulate interaction of arrestin with phosphorylation-deficient GPCRs in a receptor-specific manner.

cell biology↗

bioRxiv2022

Hexokinase 1b is a novel target for non-small-cell lung cancer

Deregulation of glycolysis is common in non-small cell lung cancer (NSCLC). Hexokinase (HK) enzymes catalyze the phosphoryl-group-transfer in glucose metabolism. There are a very few studies that have begun to reveal the connections between glucose metabolism and splicing programs. Unlike HK2 gene, which is expressed as a single transcript, there are several transcripts of the HK1 gene due to alternative splicing. However, the functional differential roles of HK1 isoforms in glucose metabolism and tumor progression are still elusive. Here, we show that primary NSCLC patient tumor cells metabolically differ from the normal lung epithelium where they display predominant expression of one of the HK1 transcripts, hexokinase1b (HK1b). We utilized CRISPR-Cas9 system to selectively target specific HK1b isoform in NSCLC and show that silencing HK1b in NSCLC cells inhibits tumorigenesis through diminishing glycolysis and proliferation. Our findings constitute the first demonstration of the first biochemical distinction between the HK1 splice variants. Finally, HK1b deletion sensitizes NSCLC cells to standard-of-care, cisplatin treatment, and the combination therapy synergistically increases both apoptotic cell death by cisplatin and autophagic cell death by increased formation of LC3-II associated autophagic vesicles and myelinoid bodies. Notably, loss of HK1b leads to cellular DNA damage, further combination with cisplatin therapy showed significantly increased levels of DNA damage. Importantly, we showed that glycolysis and cisplatin resistance can be restored by adding-back HK1b in HK1b knock-out cells. Our findings reveal that targeting HK1b isoform alone or in combination with cisplatin may represent a novel strategy for NSCLC patients.

cancer biology↗

bioRxiv2022

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

cancer biology↗