Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA ...polymerases are thought to form a complex at the stalled replication fork that we refer to as the mutasome. This hypothetical multi-protein complex is structurally organized by the replication accessory factor PCNA and the non-classical polymerase Rev1. One of the non-classical polymerases within this complex then catalyzes replication through the damage. Each non-classical polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical polymerase is chosen to bypass the damage. In this review article, we discuss how the most appropriate polymerase is chosen. In so doing, we examine the structural motifs that mediate the protein interactions in the mutasome; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
•Collided ribosomes form a unique disomal interface and are a hallmark of slow/stalled translation.•Ribosome ubiquitination is triggered by ribosome collisions targeting proteins at the disome ...interface.•Ribosome ubiquitination is linked to No-Go/ Non-Stop mRNA decay and to ribosome-associated quality control.•SMG1-8-9 structures and biochemistry indicate that GTP binding/hydrolysis by SMG9 regulates SMG1 activity.•SMG8 conformational rearrangements could limit access to SMG1’s kinase active site regulating UPF1 phosphorylation, a key step in NMD.
Eukaryotes possess a variety of translational control mechanisms which function in the surveillance of mRNAs, discriminating between normal and aberrant translation elongation and termination, triggering mRNA decay. The three major evolutionarily conserved eukaryotic pathways are No-Go, Non-Stop and Nonsense-Mediated mRNA Decay. Recent findings suggest that stalling of the ribosome, due to mRNA secondary structure or translation into poly(A)-stretches, leads to ribosome collisions which are detected by No-Go/Non-Stop mRNA decay factors. Subsequent ribosome ubiquitination at the interface of two collided ribosomes is considered the signal for mRNA decay. Similarly, translation termination at a premature stop codon is slower than normal, leading to recruitment and activation of nonsense-mediated mRNA decay factors, including SMG1-8-9. Here, we detail new insights into the molecular mechanisms of these pathways.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required ...for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches-including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering-we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.
Abstract
UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are ...differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.
Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins ...suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.
Display omitted
•Ubiquitin-modified and SUMO-modified PCNAs regulate DNA damage tolerance pathways.•We used full-ensemble methods to model the flexibility of modified PCNA proteins.•Ubiquitin and SUMO occupy many positions around the back and side of the PCNA ring.•There are no preferred ubiquitin or SUMO-binding sites on the surface of PCNA.•Flexibility facilitates effector protein recognition by modified PCNA proteins.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Translesion synthesis is the process by which nonclassical DNA polymerases bypass DNA damage during DNA replication. Cells possess a variety of nonclassical polymerases, each one is specific for ...incorporating nucleotides opposite to one or more closely related DNA lesions, called its cognate lesions. In this chapter, we discuss a variety of approaches for probing the catalytic activities and the protein-protein interactions of nonclassical polymerases. With respect to their catalytic activities, we discuss polymerase assays, steady-state kinetics, and presteady-state kinetics. With respect to their interactions, we discuss qualitative binding assays such as enzyme-linked immunosorbent assays and coimmunoprecipitation; quantitative binding assays such as isothermal titration calorimetry, surface plasmon resonance, and nuclear magnetic resonance spectroscopy; and single-molecule binding assays such as total internal reflection fluorescence microscopy. We focus on how nonclassical polymerases accommodate their cognate lesions during nucleotide incorporation and how the most appropriate nonclassical polymerase is selected for bypassing a given lesion.
Y-family DNA polymerases, such as polymerase η, polymerase ι, and polymerase κ, catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by ...interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase η, a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase η mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase η binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase κ and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation ...by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
Background:
Extreme conditioning programs (ECPs) are fitness training regimens relying on aerobic, plyometric, and resistance training exercises, often with high levels of intensity for a short ...duration of time. These programs have grown rapidly in popularity in recent years, but science describing the safety profile of these programs is lacking.
Hypothesis:
The rate of injury in the extreme conditioning program is greater than the injury rate of weightlifting and the majority of injuries occur to the shoulder and back.
Study Design:
Cross-sectional study.
Level of Evidence:
Level 4.
Methods:
This is a retrospective survey of injuries reported by athletes participating in an ECP. An injury survey was sent to 1100 members of Iron Tribe Fitness, a gym franchise with 5 locations across Birmingham, Alabama, that employs exercises consistent with an ECP in this study. An injury was defined as a physical condition resulting from ECP participation that caused the athlete to either seek medical treatment, take time off from exercising, or make modifications to his or her technique to continue.
Results:
A total of 247 athletes (22%) completed the survey. The majority (57%) of athletes were male (n = 139), and 94% of athletes were white (n = 227). The mean age of athletes was 38.9 years (±8.9 years). Athletes reported participation in the ECP for, on average, 3.6 hours per week (± 1.2 hours). Eighty-five athletes (34%) reported that they had sustained an injury while participating in the ECP. A total of 132 injuries were recorded, yielding an estimated incidence of 2.71 per 1000 hours. The shoulder or upper arm was the most commonly injured body site, accounting for 38 injuries (15% of athletes). Athletes with a previous shoulder injury were 8.1 times as likely to injure their shoulder in the ECP compared with athletes with healthy shoulders. The trunk, back, head, or neck (n = 29, 12%) and the leg or knee (n = 29, 12%) were the second most commonly injured sites. The injury incidence rate among athletes with <6 months of experience in the ECP was 2.5 times greater than that of more experienced athletes (≥6 months of experience). Of the 132 injuries, 23 (17%) required surgical intervention. Squat cleans, ring dips, overhead squats, and push presses were more likely to cause injury. Athletes reported that 35% of injuries were due to overexertion and 20% were due to improper technique.
Conclusion:
The estimated injury rate among athletes participating in this ECP was similar to the rate of injury in weightlifting and most other recreational activities. The shoulder or upper arm was the most commonly injured area, and previous shoulder injury predisposed to new shoulder injury. New athletes are at considerable risk of injury compared with more experienced athletes.
Clinical Relevance:
Extreme conditioning programs are growing in popularity, and there is disagreement between science and anecdotal reports from athletes, coaches, and physicians about their relative safety. This study estimates the incidence of injury in extreme conditioning programs, which appears to be similar to other weight-training programs.
Full text
Available for:
FSPLJ, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal ...transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.
Display omitted
•Conformationally flexible proteins are a major challenge for structural biologists.•Flexible proteins can be examined by combining molecular simulations and SAXS.•Minimal ensemble searches are a common way of combining simulations and SAXS.•Full ensemble methods use SAXS to validate simulations without curve fitting.•Full ensemble models are more realistic than minimal ensemble searches models.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
