Biology subjects

Wanrooij, S.

Publications and source records attributed to Wanrooij, S..

10 recordsLinked to original sources

bioRxiv2026

Dual Recognition Drives Site-Directed G-Quadruplex Stabilization: Exploring Oligonucleotide Design in G4 Ligand-Oligonucleotide Conjugates

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in key cellular processes, including transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among human G4 motifs. The G4 Ligand-conjugated Oligonucleotide strategy addresses this need by combining the G4-binding capabilities of small-molecule G4-ligands with the sequence specificity of an oligonucleotide complementary to the flanking region of the target G4. Here, we systematically explore how the oligonucleotide component governs G4 binding and stabilization by varying its length, backbone composition, and sequence complementarity. This revealed that efficient G4 recognition depends on a strong interdependence between hybridization and G4-ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Central mismatches disrupt this dual recognition and reduce selectivity. While longer oligonucleotides hybridize more slowly, they form more stable complexes and show stronger G4 stabilization in thermal melting and polymerase stop assays. Replacing the DNA oligonucleotide with peptide nucleic acid enhances binding strength, thermal stability, and metabolic stability, but selective G4 stabilization is achieved only upon ligand conjugation. Together, these results show how rational oligonucleotide design enables selective and potent recognition of G4 structures using GL-Os.

molecular biology↗

bioRxiv2026

Redox-dependent dimerization of PolDIP2 and a conserved ApaG-domain motif required for CHCHD2 interaction

PolDIP2 is a multifunctional mitochondrial protein implicated in redox regulation, mitochondrial proteostasis, and diverse mtDNA-associated processes, yet the principles underlying its regulation remain unclear. Crystallographic analysis revealed that PolDIP2 forms a redox-dependent disulfide-linked homodimer via a conserved Cys143 residue within its N-terminal YccV-like domain, and cellular and in vitro assays confirmed that this residue is essential for dimer formation. Oxidative stress enhanced dimerization of endogenous and ectopically expressed PolDIP2, and dimers were detected exclusively within mitochondria, requiring proper mitochondrial import. WT and C143A PolDIP2 overexpression produced similarly modest effects on mtDNA replication in cells, suggesting that dimerization has limited impact on mtDNA-associated processes. Proteomic analysis and biochemical validation identified both previously known and not yet characterized mitochondrial interactors of PolDIP2, and highlighted CHCHD2 as a specific binding partner. A conserved glycine-rich motif in the C-terminal ApaG/DUF525-like domain proved essential for this interaction, and disruption of the motif enhanced Cys143-dependent dimerization while abolishing CHCHD2 association, which preferentially occurs with monomeric PolDIP2. These findings define redox-controlled dimerization and a conserved ApaG-domain motif as key structural features shaping PolDIP2s interaction state within mitochondria and provide a basis for exploring its roles in redox-sensitive mitochondrial pathways.

cell biology↗

bioRxiv2024

The Y951N patient mutation inactivates the intramolecular switch in human mitochondrial DNA POLgamma

Mitochondrial DNA (mtDNA) stability, essential for cellular energy production, relies on DNA polymerase gamma (POL{gamma}). Here, we show that the POL{gamma} Y951N disease causing mutation induces replication stalling and severe mtDNA depletion. However, unlike other POL{gamma} disease causing mutations, Y951N does not directly impair exonuclease activity and only mildly affects polymerase activity. Instead, we found that Y951N compromises the enzymes ability to efficiently toggle between DNA synthesis and degradation, and is thus the first patient-derived mutation with impaired polymerase-exonuclease switching. These findings provide new insights into the intramolecular switch when POL{gamma} proofreads the newly-synthesized DNA strand, and reveal a new mechanism for causing mitochondrial DNA instability. Significance StatementDNA polymerase gamma (POL{gamma}) is essential for copying mitochondrial DNA (mtDNA), which is crucial for our energy production. POL{gamma} must accurately switch between making new DNA (polymerase activity) and correcting errors (exonuclease activity). While it is known that mutations in POL{gamma} can cause mitochondrial diseases by directly impairing these enzymatic functions, this study reveals a new mechanism. The Y951N mutation disrupts POL{gamma}s ability to switch between these activities, leading to severe blockages in DNA replication and a loss of mtDNA in human cells, even without significant direct impairment of polymerase or exonuclease activities. These findings provide new insights into the origins of mitochondrial diseases.

biochemistry↗

bioRxiv2024

Preferential binding of ADP-bound mitochondrial HSP70 to the nucleotide exchange factor GRPEL1 over GRPEL2

Human nucleotide exchange factors GRPEL1 and GRPEL2 play pivotal roles in the ADP-ATP exchange within the protein folding cycle of mitochondrial HSP70 (mtHSP70), a crucial chaperone facilitating protein import into the mitochondrial matrix. Studies in human cells and mice have indicated that while GRPEL1 serves as an essential co-chaperone for mtHSP70, GRPEL2 has a role regulated by stress. However, the precise structural and biochemical mechanisms underlying the distinct functions of the GRPEL proteins have remained elusive. In our study, we present evidence revealing that ADP-bound mtHSP70 exhibits remarkably higher affinity for GRPEL1 compared to GRPEL2, with the latter experiencing a notable decrease in affinity upon ADP binding. Utilizing Alphafold modelling, we propose that the interaction between GRPEL1 and mtHSP70 can induce the opening of the nucleotide binding cleft of the chaperone, thereby facilitating the release of ADP, whereas GRPEL2 lacks this capability. This study elucidates how ADP binding to mtHSP70 enhances its affinity for GRPEL1, contrasting with its interaction with GRPEL2. Additionally, our findings suggest that the redox-regulated Cys87 residue in GRPEL2 does not play a role in dimerization but rather reduces its affinity for mtHSP70. Our findings on the structural and functional disparities between GRPEL1 and GRPEL2 may have implications for mitochondrial protein folding and import processes under varying cellular conditions.

biochemistry↗

bioRxiv2024

The specific AMPK activator A-769662 ameliorates pathological phenotypes following mitochondrial DNA depletion

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis that also plays a role in preserving mitochondrial function and integrity. Upon a disturbance in the cellular energy state that increases AMP levels, AMPK activity promotes a switch from anabolic to catabolic metabolism to restore energy homeostasis. However, it is currently unclear how severe of a mitochondrial dysfunction is required to trigger AMPK activation, and whether stimulation of AMPK using specific agonists can improve the cellular phenotype following mitochondrial dysfunction. Using a cell model of mitochondrial disease characterized by progressive mitochondrial DNA (mtDNA) depletion and deteriorating mitochondrial metabolism, we show that mitochondria-associated AMPK becomes activated early in the course of the advancing mitochondrial dysfunction, before any quantifiable decrease in the ATP/(AMP+ADP) ratio or respiratory chain activity. Moreover, stimulation of AMPK activity using the specific small-molecule agonist A-769662 alleviated the mitochondrial phenotypes caused by the mtDNA depletion and restored normal mitochondrial membrane potential. Notably, the agonist treatment was able to partially restore mtDNA levels in cells with severe mtDNA depletion, while it had no impact on mtDNA levels of control cells. The beneficial impact of the agonist was also observed in cells from patients suffering from mtDNA depletion. However, the positive effects of A-769662 in the two experimental cell models appeared to involve at least partially different mechanisms. These findings improve our understanding of the effects of specific small-molecule activators of AMPK on mitochondrial and cellular function, and suggest a potential utility for these compounds in disease states involving mtDNA depletion.

molecular biology↗

bioRxiv2024

A small molecule inhibitor Mirin prevents TOP3A-dependent mtDNA breakage and segregation

Mirin, the chemical inhibitor of MRE11, has been recently reported to prevent immune response activation caused by mitochondrial DNA (mtDNA) breakage and release upon replication stalling. We show here that Mirin prevents mitochondrial replication fork breakage in mitochondrial 3-exonuclease MGME1 deficient cells and the resulting innate immune response induction, but that this occurs independently of MRE11. Furthermore, Mirin also caused alteration of mtDNA supercoiling and accumulation of hemicatenated replication termination intermediates, hallmarks of topoisomerase dysfunction, as well as alleviated topological changes induced by the overexpression of mitochondrial TOP3A, including TOP3A-dependent strand breakage at the non-coding region of mtDNA, potentially explaining its protective effect in the MGME1-knockout cells. Although Mirin does not inhibit TOP3A in vitro, our results demonstrate its MRE11-independent effects in cells and give insight into the mechanisms of mtDNA segregation, as well as the maintenance of genomic integrity in mitochondria. Significance StatementO_LIBroken mitochondrial DNA (mtDNA) in MGME1 knockout cells activates innate immune response, which is prevented by Mirin, a small molecule inhibitor of MRE11. C_LIO_LIMirin also interferes with mtDNA replication termination and segregation, suggesting that termination intermediates or paused forks are a major source of mtDNA breakage. C_LIO_LIWe show that these effects are likely dependent on topoisomerase 3A (TOP3A) -related processes in mitochondria, questioning the Mirin target also in the nucleus. C_LI

molecular biology↗

bioRxiv2023

G4-Ligand Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a longstanding problem in the field is how to achieve specificity amongst these different G4 structures. Here, we have developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4 Ligand conjugated Oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amounts of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

biochemistry↗

bioRxiv2022

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrated that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

cell biology↗

bioRxiv2021

Towards customized studies of G-quadruplex DNA structures in live cells

G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. To evaluate the power of the approach, we next used this reactive handle to conjugate a BODIPY fluorophore and a cell-penetrating peptide to Phen-DC3. The BODIPY conjugation generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effects that revealed the localization and function of Phen-DC3 in human cells. On the other hand, the cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this approach to modulate and direct cellular uptake e.g. as delivery vehicles. The developed platform can thus generate tailored biochemical tools for the studies of G4 biology to uncover molecular details and therapeutic approaches.

cell biology↗

bioRxiv2020

A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

Replication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Pol{lambda}, Pol{eta} and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPols enhanced processivity. PolDIP2 increases PrimPols primer-template and dNTP binding affinity, which concomitantly enhances PrimPols nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Pol{lambda}, Pol{eta}, Pol{zeta} and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.

biochemistry↗