Sliding clamps are ring-shaped protein complexes that integral to the DNA replication machinery of all life. opened and installed onto by clamp loader AAA+ ATPase complexes. However, how a opens closes sliding around is still unknown. Here, we describe structures Saccharomyces cerevisiae Replication Factor C (RFC) bound its cognate Proliferating Cell Nuclear Antigen (PCNA) en route successful loading. RFC first binds PCNA in dynamic, closed conformation blocks both activity binding. then...

A virally encoded ASCE ATPase motor ratchets between helical and planar configurations to translocate dsDNA into viral procapsids.

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

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 force measurements on the packaging motor of bacteriophage φ29 revealed that its five ATPase subunits coordinate ATP hydrolysis with each other to maintain proper cyclic sequence translocation steps about ring. Here, we explore how regulates by timing key events, namely binding/hydrolysis gripping,...

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

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 ring-shaped subsequent binding to primer-template (p/t)-junctions. general architecture conserved across all life, suggesting their mechanism retained. Recent structural studies eukaryotic factor C (RFC) revealed it functions using crab-claw mechanism, coupled massive conformational change loader....

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These have conserved Walker A B motifs that bind Mg2+-ATP a catalytic carboxylate activates water for hydrolysis. Here we demonstrate Glu179 serves the in bacteriophage λ terminase probe its mechanistic role. All changes of are lethal: non-conservative abrogate ATP hydrolysis translocation, while conservative E179D change attenuates alters single molecule translocation dynamics,...

Significance Many biological processes are performed by ring-shaped molecular motors that harvest energy from ATP hydrolysis to move DNA, RNA, or protein substrate through their central pores. However, it is not clear how these proteins couple physical movement the chemical steps of hydrolysis. Here, we derive mechanism which viral DNA packaging binding gripping and release. We first predict coupling pathway computationally with dynamics simulations then validate our predictions...

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner can synthesize, scan, and repair the genome. with nicks or small single-stranded gaps are common clamp-loading targets in repair, yet these substrates would be sterically blocked given known mechanism for binding of primer-template DNA. Here, we report discovery a second site yeast loader replication factor C (RFC) aids to nicked gapped This is on external surface only accessible open conformation RFC....

Tail tube assembly is an essential step in the lifecycle of long-tailed bacteriophages. Limited structural and biophysical information has impeded understanding stability their long, flexible tail tubes. The hyperthermophilic phage P74-26 particularly intriguing as it longest any known virus (nearly 1 μm) most thermostable phage. Here, we use structures along with vitro system for studying kinetics to propose first molecular model phages. Our high-resolution cryo-EM structure provides...

SUMMARY Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces macromolecules. The additional strand conserved E (ASCE) ring are an ancient family responsible for diverse tasks ranging from biological polymer (e.g. protein degradation chromosome segregation) establishing maintaining proton gradients across mitochondrial membranes. Viruses also utilize ASCE package their genomes into capsids serve as accessible...

Summary 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 form viral dsDNA packaging motor and characterize its atomic-level dynamics via long timescale molecular simulations. Based on results, we deduce an overall mechanism that is driven by helical-to-planar transitions motor. These are coordinated inter-subunit interactions regulate catalytic...

Abstract The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated translocation in by performing long timescale simulations of its hexameric assembly and individual subunits. From these predict signaling pathways that connect ATPase active site DNA-gripping residues, which allows coordinate activity with activity. Additionally, utilize well-tempered metadynamics compute...

Abstract 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 yeast cells exposed to oxidative stress, stalling ribosomes at elongation. conjugates accumulate because of redox inhibition the deubiquitinating enzyme Ubp2, however, role regulation conjugating enzymes this pathway remained unclear. Here we found E2 Rad6 binds modifies elongating during...

SUMMARY Tail tube assembly is an essential step in the of long-tailed bacteriophages. Limited structural and biophysical information has impeded understanding stability their long, flexible tail tubes. The hyperthermophilic phage P74-26 particularly intriguing as it longest any known virus (nearly 1 μm) most stable phage. Here, we present structure introduce vitro system for studying kinetics assembly. Our high resolution cryo-EM provides insight into how achieves its flexibility...

SUMMARY Sliding clamps are ring-shaped protein complexes that integral to the DNA replication machinery of all life. opened and installed onto by clamp loader AAA+ ATPase complexes. However, how a opens closes sliding around is still unknown. Here, we describe structures S. cerevisiae Replication Factor C (RFC) bound its cognate Proliferating Cell Nuclear Antigen (PCNA) en route successful loading. RFC first binds PCNA in dynamic, closed conformation blocks both activity binding. then ring...

SUMMARY 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 ring-shaped clamp, subsequent binding to primer-template (p/t)-junctions. general architecture conserved across all life, suggesting their mechanism retained. Recent structural studies eukaryotic Replication Factor C (RFC) revealed it functions using crab-claw mechanism, coupled massive...