The mechano-chemistry of a viral genome packaging motor.

Double-stranded DNA viruses actively package their genomes into pre-assembled protein capsids using energy derived from virus-encoded ASCE ATPase ring motors. Single molecule experiments in the aughts and early 2010s demonstrated that these motors are some of the most powerful molecular motors in nature, and that the activities of individual subunits around the ATPase ring motor are highly coordinated to ensure efficient genome encapsidation. While these studies provided a comprehensive kinetic scheme describing the events that occur during packaging, the physical basis of force generation and subunit coordination remained elusive.

Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication.

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.

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.

Conformational dynamics control assembly of an extremely long bacteriophage tail tube.

Tail tube assembly is an essential step in the lifecycle 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 μm) and is the most thermostable known phage.

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.

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.

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

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear.

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.

Atomistic basis 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, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor.

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 biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity.

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.

Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif.

Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >10-fold decrease in phage yield, but we identified nine mutants with partial activity.

Evidence that a catalytic glutamate and an 'Arginine Toggle' act in concert to mediate ATP hydrolysis and mechanochemical coupling in a viral DNA packaging motor.

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role.