Biology subjects

Pajak, J.

Publications and source records attributed to Pajak, J..

11 recordsLinked to original sources

bioRxiv2025

PCNA is a Nucleotide Exchange Factor for the Clamp Loader ATPase Complex

All life requires loading ring-shaped sliding clamp protein complexes onto DNA. The sliding clamp loader is a conserved AAA+ ATPase that binds the sliding clamp, opens the ring, and places it onto DNA. While recent structural work on both the canonical and alternative clamp loaders has shed light into how these machines perform their task once, it remains unclear how clamp loaders are recycled to load multiple sliding clamps. Here, we present structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) in absence of sliding clamp or supplemented nucleotide. Our structures indicate that RFC holds onto ADP tightly in at least two of its four ATPase active sites, suggesting that nucleotide exchange is regulated. Our molecular dynamics simulations and biochemical data indicate that binding of the sliding clamp PCNA causes rapid exchange of tightly bound ADP. Our data suggests that PCNA acts as a nucleotide exchange factor by prying apart adjacent subunits, providing a pathway for ADP release. We propose that, by using its own substrate as a nucleotide exchange factor, RFC excludes off-pathway states that would arise from binding DNA prior to PCNA.

biochemistry↗

bioRxiv2025

Regulation of Ordinal DNA Translocation Cycle in Bacteriophage {varphi}29 through Trans-Subunit Interactions

Certain viruses such as tailed bacteriophages and herpes simplex virus package double-stranded DNA into empty procapsids via powerful, ring-shaped molecular motors. High resolution structures and force measurements on the DNA packaging motor of bacteriophage {Phi}29 revealed that its five ATPase subunits coordinate ATP hydrolysis with each other to maintain the proper cyclic sequence of DNA translocation steps about the ring. Here, we explore how the {Phi}29 motor regulates translocation by timing key events, namely ATP binding/hydrolysis and DNA gripping, through trans-subunit interactions. We used subunit dimers bound to DNA as our model system, a minimal system that still captures the conformation and trans-subunit interactions of the full pentametric motor complex. Molecular dynamics simulations of all-ATP and mixed ATP-ADP dimers revealed that the nucleotide occupancy of one subunit strongly affects the ability to hydrolyze ATP in the adjacent subunit by altering the free energy landscape of its catalytic glutamate approaching the gamma phosphate of ATP. Specifically, one ATP-bound subunit donates residues in trans that sterically block the neighboring subunits catalytic glutamate. This steric hindrance is resolved when the first subunit hydrolyzes ATP and is ADP-bound. This obstructive mechanism is supported by functional mutagenesis and appears to be conserved across several {Phi}29 relatives. Mutual information analysis of our simulations revealed intersubunit signaling pathways, via the trans-acting obstructive residues, that allow for sensing and communication between the binding pockets of adjacent subunits. This work shows that the sequential order of DNA translocation events amongst subunits is preserved through novel trans-subunit interactions and pathways.

molecular biology↗

bioRxiv2023

Differences in clamp loader mechanism between bacteria and eukaryotes

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp, and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader Replication Factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the E. coli clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

biochemistry↗

bioRxiv2022

Structure and assembly of an extremely long bacteriophage tail tube

Tail tube assembly is an essential step in the assembly of long-tailed bacteriophages. Limited structural and biophysical information has impeded an understanding of assembly and stability of their long, flexible tail tubes. The hyperthermophilic phage P74-26 is particularly intriguing as it has the longest tail of any known virus (nearly 1 m) and is the most stable known phage. Here, we present the structure of the P74-26 tail tube and introduce an in vitro system for studying the kinetics of tube assembly. Our high resolution cryo-EM structure provides insight into how the P74-26 phage achieves its flexibility and thermostability through assembly of flexible loops into neighboring rings through tight "ball-and-socket"-like interactions. Guided by this structure, and in combination with mutational, light scattering, and molecular dynamics simulations data, we propose a model for the assembly of conserved tube-like structures across phage and other entities possessing Tail Tube-like proteins. Our model proposes that formation of a full ring licenses the adoption of a tube elongation-competent conformation among the flexible loops and their corresponding sockets, which is further stabilized by an adjacent ring. Tail assembly is controlled by the cooperative interaction of dynamic intra- and inter-ring contacts. Given the structural conservation among tail tube proteins and tail-like structures, our model can explain the mechanism of high-fidelity assembly of long, stable tubes.

biophysics↗

bioRxiv2022

A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFCs central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFCs external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.

biochemistry↗

bioRxiv2022

Molecular Dynamics of DNA Translocation by FtsK

The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.

biophysics↗

bioRxiv2021

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale crab-claw expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFCs switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

biochemistry↗

bioRxiv2021

Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes.

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63 ubiquitin conjugates accumulate because of redox inhibition of the deubiquitinating enzyme Ubp2, however, the role and regulation of ubiquitin conjugating enzymes in this pathway remained unclear. Here we found that the E2 Rad6 binds and modifies elongating ribosomes during oxidative stress. We elucidated a mechanism by which Rad6 and its human homolog UBE2A are redox-regulated by forming reversible disulfides with the E1 activating enzyme, Uba1. We further showed that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress. Finally, we showed that Rad6 is required to induce phosphorylation of the translation initiation factor eIF2, providing a novel link for K63 ubiquitin, elongation stalling, and the integrated stress response.

cell biology↗

bioRxiv2020

Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the {varphi}29 DNA packaging motor. Our work thus establishes a signaling pathway in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging.

biophysics↗

bioRxiv2020

Atomistic Mechanism of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on the results, we deduce an overall packaging mechanism that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases that exhibit burst-dwell dynamics.

molecular biology↗

bioRxiv2020

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse tasks ranging from biological polymer manipulation (e.g. protein degradation and chromosome segregation) to establishing and maintaining proton gradients across mitochondrial membranes. Viruses also utilize ASCE segregation motors to package their genomes into their protein capsids and serve as accessible experimental systems due to their relative simplicity. We show by CryoEM focused image reconstruction that ASCE ATPases in viral dsDNA packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, including structural results showing these ATPases in planar ring conformations, our results suggest that these motors cycle between helical and planar cyclical symmetry, providing a possible mechanism for directional translocation of DNA. We further note that similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

biophysics↗