Ariel Kaplan | Technion - Israel Institute of Technology (original) (raw)

Papers by Ariel Kaplan

Nature Communications

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding ... more The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s−1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies — including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry — we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

eLife, 2019

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA ... more The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD’s individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25–40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

Biophysical Journal, 2017

polymerase in E. coli. It can synthesize DNA at a rate of 1000 bp/s. During proofreading, the pri... more polymerase in E. coli. It can synthesize DNA at a rate of 1000 bp/s. During proofreading, the primer strand migrates from the polymerase domain to the exonuclease domain, a distance of 60 Å for DNA Pol III. As the dynamics of this process is not well understood, the goal of this work is to evaluate active site switching dynamics during proofreading at a single-molecule level. For this, the catalytic core of the DNA Pol III holoenzyme comprising the polymerase a subunit, the sliding clamp b 2 subunit, the exonuclease ε subunit and a q subunit was tested. We also examined proofreading with a and ε subunit mutants with altered exonuclease activity. In this study, active site switching was monitored with varying errors on primer-template termini including several mismatches and an abasic site analog paired opposite A. Single-molecule FRET data will be presented that measured the kinetics of primer strand transfer as well as the distribution of the DNA in the polymerase and exonuclease domains. This, along with primer extension assays performed in bulk, reveal that the dynamics of proofreading vary as a function of the terminal DNA mismatch. Also, single-molecule protein induced fluorescence assays will be presented for the binding kinetics of the a and aε subunits. Overall, our work provides unique insight into the mechanism that ensures high fidelity DNA synthesis and preserves the integrity of the information stored in the genomic DNA.

Nature Communications, 2016

The structure and dynamics of promoter chromatin have a profound effect on the expression levels ... more The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes, Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (þ 1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.

Nucleic Acids Research, 2021

The interaction of transcription factors with their response elements in DNA is emerging as a hig... more The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein&#39...

We developed a new experimental system (the “atom-optics billiard”) and demonstrated chaotic and ... more We developed a new experimental system (the “atom-optics billiard”) and demonstrated chaotic and regular dynamics of cold, optically trapped atoms. We show that the softness of the walls and additional optical potentials can be used to manipulate the structure of phase space.

Physical review letters, 2001

Proceedings of the National Academy of Sciences

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind... more The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone β subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF’s binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as...

arXiv: Chaotic Dynamics, 2004

We present a new experimental system (the ``atom-optics billiard'') and demonstrate chaot... more We present a new experimental system (the ``atom-optics billiard'') and demonstrate chaotic and regular dynamics of cold, optically trapped atoms. We show that the softness of the walls and additional optical potentials can be used to manipulate the structure of phase space.

Nature Reviews Endocrinology

RecBCD, responsible for the initiation of double stranded break repair in bacteria, is a processi... more RecBCD, responsible for the initiation of double stranded break repair in bacteria, is a processive DNA helicase with an unwinding rate approaching ~1,600 bp s -1 . The mechanism enabling RecBCD to achieve such fast unwinding rate is not known. We employed a combination of equilibrium and time-resolved binding experiments, and ensemble and single molecule activity assays to uncover the molecular mechanism underlying RecBCD's rapid catalysis. We report the existence of auxiliary binding sites, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. The catalytic rate of RecBCD is reduced both by preventing and by strengthening ATP binding to these sites, suggesting that the dynamics of ATP at these sites modulates the enzyme's rate. We propose a model by which RecBCD achieves its fast unwinding rate by utilizing the weaker binding sites to increase the flux of ATP to its catalytic sites.

Frontiers in Endocrinology

Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitar... more Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

Cell Death & Disease

Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysoso... more Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca 2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca 2+ , or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.

Nucleic acids research, Jan 15, 2017

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecu... more Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

Nucleic Acids Research

Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competen... more Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a nontemplate strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.

Nucleosomes, the basic building block of chromatin, regulate the accessibility of the transcripti... more Nucleosomes, the basic building block of chromatin, regulate the accessibility of the transcription machinery to DNA. Recent studies have revealed that the nucleosome's spontaneous, thermally driven positional dynamics are modulated by different factors, and exploited by the cell as a regulatory mechanism. In particular, enrichment of mobile nucleosomes at the promoters of genes suggests that the mobility of nucleosomes may affect the ability of transcription factors to bind DNA. However, a quantitative model describing the effect nucleosome mobility on the effective affinity of transcription factors is lacking. We present here a simple equilibrium model that captures the essence of the effect, and show that modulation of the nucleosome's mobility can be a potent and versatile regulator of transcription factor binding.