Visualizing Rev1 catalyze protein-template DNA synthesis.

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

Engagement of DNA and H3K27me3 by the CBX8 chromodomain drives chromatin association.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones. For CBX8, however, the chromodomain has demonstrated weak affinity and specificity for methylated histones in vitro, leaving doubt as to its role in CBX8 chromatin association. Here, we investigate the function of the CBX8 CD in vitro and in vivo. We find that the CD is in fact a major driver of CBX8 chromatin association and determine that this is driven by both histone and previously unrecognized DNA binding activity. We characterize the structural basis of histone and DNA binding and determine how they integrate on multiple levels. Notably, we find that the chromatin environment is critical in determining the ultimate function of the CD in CBX8 association.

Manual patient transfers: factors that influence decisions and kinematic strategies employed by nursing aides.

While extensive literature has characterised factors that influence the acceptable mass of 'boxes' during MMH tasks, less is known about these factors when moving 'people' in healthcare settings. This study examined factors that influence decisions/approaches employed during manual patient transfers. Sixteen nursing aides manually-transferred a standardised 'patient'; patient mass was adjusted (using a weight vest) to determine a maximum acceptable patient mass for this task (massmax). Grip strength was the only worker characteristic significantly associated with massmax (r = 0.48). Older worker age was associated with smaller peak trunk flexion (r = -0.58) and shoulder abduction (r = -0.59), and greater trunk axial twist (r = 0.52). Workers emphasised that patient characteristics (e.g. physical/cognitive status) influenced their decisions when performing transfers. These findings extend previous literature by suggesting that grip strength is a useful predictor of perceived work capacity, older workers adapt protective postural strategies during patient transfers and worker-patient dynamics are crucial during this high-risk occupational task. Practitioner Summary: This study examined manual patient transfers performed by nursing aides. Worker grip strength (but not age or size) was associated with perceptions of maximum acceptable patient mass. Kinematic changes suggested more conservative strategies used by older workers. Workers emphasised that patient characteristics substantially influenced their decisions when performing transfer tasks.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli.

Reading More than Histones: The Prevalence of Nucleic Acid Binding among Reader Domains.

The eukaryotic genome is packaged into the cell nucleus in the form of chromatin, a complex of genomic DNA and histone proteins. Chromatin structure regulation is critical for all DNA templated processes and involves, among many things, extensive post-translational modification of the histone proteins. These modifications can be "read out" by histone binding subdomains known as histone reader domains. A large number of reader domains have been identified and found to selectively recognize an array of histone post-translational modifications in order to target, retain, or regulate chromatin-modifying and remodeling complexes at their substrates. Interestingly, an increasing number of these histone reader domains are being identified as also harboring nucleic acid binding activity. In this review, we present a summary of the histone reader domains currently known to bind nucleic acids, with a focus on the molecular mechanisms of binding and the interplay between DNA and histone recognition. Additionally, we highlight the functional implications of nucleic acid binding in chromatin association and regulation. We propose that nucleic acid binding is as functionally important as histone binding, and that a significant portion of the as yet untested reader domains will emerge to have nucleic acid binding capabilities.

Standing Versus Stepping-Exploring the Relationships Between Postural Steadiness and Dynamic Reactive Balance Control.

While the literature has characterized balance control during quasi-static and/or dynamic tasks, comparatively few studies have examined relationships across paradigms. This study investigated whether quiet-stance postural steadiness metrics were associated with reactive control parameters (during both stepping and restabilization phases) following a lean-and-release perturbation. A total of 40 older adults participated. Postural steadiness (center of the pressure range, root mean square, velocity, and frequency) was evaluated in "feet together" and "tandem stance" positions. During the reactive control trials, the step length, step width, movement time, and reaction time were measured, in addition to the postural steadiness variables measured during the restabilization phase following the stepping response. Out of 64 comparisons, only 10 moderate correlations were observed between postural steadiness and reactive spatio-temporal stepping parameters (P ≤ .05, r = -.312 to -.534). However, postural steadiness metrics were associated with the center of pressure velocity and frequency during the restabilization phase of the reactive control trials (P ≤ .02, r = .383 to .775 for velocity and P ≤ .01, r = .386 to .550 for frequency). Although some elements of quasi-static center of pressure control demonstrated moderate associations with dynamic stepping responses, relationships were stronger for restabilization phase dynamics after foot-contact. Future work should examine the potential association between restabilization phase control and older adult fall-risk.

Use of the Nintendo Wii Balance Board for Studying Standing Static Balance Control: Technical Considerations, Force-Plate Congruency, and the Effect of Battery Life.

The Nintendo Wii Balance Board (WBB) has become popular as a low-cost alternative to research-grade force plates. The purposes of this study were to characterize a series of technical specifications for the WBB, to compare balance control metrics derived from time-varying center of pressure (COP) signals collected simultaneously from a WBB and a research-grade force plate, and to investigate the effects of battery life. Drift, linearity, hysteresis, mass accuracy, uniformity of response, and COP accuracy were assessed from a WBB. In addition, 6 participants completed an eyes-closed quiet standing task on the WBB (at 3 battery life levels) mounted on a force plate while sway was simultaneously measured by both systems. Characterization results were all associated with less than 1% error. R2 values reflecting WBB sensor linearity were > .99. Known and measured COP differences were lowest at the center of the WBB and greatest at the corners. Between-device differences in quiet stance COP summary metrics were of limited clinical significance. Lastly, battery life did not affect WBB COP accuracy, but did influence 2 of 8 quiet stance WBB parameters. This study provides general support for the WBB as a low-cost alternative to research-grade force plates for quantifying COP movement during standing.

Single-molecule analysis of purified proteins and nuclear extracts: Insights from 8-oxoguanine glycosylase 1.

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Nucleolytic processing of abasic sites underlies PARP inhibitor hypersensitivity in ALC1-deficient BRCA mutant cancer cells.

Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites. We show that ALC1 enhances the ability of the abasic site processing enzyme, Apurinic/Apyrimidinic endonuclease 1 (APE1) to cleave nucleosome-occluded abasic sites. However, unrepaired abasic sites in ALC1-deficient cells are readily accessed by APE1 at the nucleosome-free replication forks. APE1 cleavage leads to fork breakage and trapping of PARP1/2 upon PARPi treatment, resulting in hypersensitivity. Collectively, our studies reveal how cells overcome the chromatin barrier to repair abasic lesions and uncover cleavage of abasic sites as a mechanism to overcome limitations of PARPi.

Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage.

Eukaryotic DNA exists in chromatin, where the genomic DNA is packaged into a fundamental repeating unit known as the nucleosome. In this chromatin environment, our genomic DNA is constantly under attack by exogenous and endogenous stressors that can lead to DNA damage. Importantly, this DNA damage must be repaired to prevent the accumulation of mutations and ensure normal cellular function. To date, most in-depth biochemical studies of DNA repair proteins have been performed in the context of free duplex DNA. However, chromatin can serve as a barrier that DNA repair enzymes must navigate in order find, access, and process DNA damage in the cell. To facilitate future studies of DNA repair in chromatin, we describe a protocol for generating nucleosome containing site-specific DNA damage that can be utilized for a variety of in vitro applications. This protocol describes several key steps including how to generate damaged DNA oligonucleotides, the expression and purification of recombinant histones, the refolding of histone complexes, and the reconstitution of nucleosomes containing site-specific DNA damage. These methods will enable researchers to generate nucleosomes containing site-specific DNA damage for extensive biochemical and structural studies of DNA repair in the nucleosome.

Single-molecule analysis of purified proteins and nuclear extracts: insights from 8-oxoguanine glycosylase 1.

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

DNA methylation plays a key role in epigenetics, with 60-80% of CpG sites containing 5-methylcytosine. Base excision repair (BER) is suggested to be the main pathway involved in active DNA demethylation. 5-formylctyosine (5fC), an oxidized moiety of methylated cytosine, is recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. TDG binds avidly to abasic sites and is product inhibited. Using single molecule fluorescence experiments, we saw TDG interact with DNA containing 5fC specifically and non-specifically with lifetimes of 72.9 and 7.5 seconds, respectively. These results indicate that TDG cleaves the 5fC and stays bound for an extended time at the generated abasic site. Mean squared displacement analysis and a two color TDG experiment indicate that TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. The catalytically crippled variants, N140A and R275A/L, have a reduced binding lifetime compared to wild type and Mean Squared Displacement (MSD) analysis indicates that R275L/A moves on the DNA with a faster diffusivity. These results indicate that mutating R275, but not N140 interferes with damage recognition by TDG. Our findings give insight into how TDG searches for its lesions in long stretches of undamaged DNA.

New insights into DNA polymerase mechanisms provided by time-lapse crystallography.

DNA polymerases play central roles in DNA replication and repair by catalyzing template-directed nucleotide incorporation. Recently time-lapse X-ray crystallography, which allows one to observe reaction intermediates, has revealed numerous and unexpected mechanistic features of DNA polymerases. In this article, we will examine recent new discoveries that have come from time-lapse crystallography that are currently transforming our understanding of the structural mechanisms used by DNA polymerases. Among these new discoveries are the binding of a third metal ion within the polymerase active site, the mechanisms of translocation along the DNA, the presence of new fidelity checkpoints, a novel pyrophosphatase activity within the active site, and the mechanisms of pyrophosphorolysis.

Anti-fatigue mats can reduce low back discomfort in transient pain developers.

Complaints of musculoskeletal pain are common among employees who stand for prolonged periods. This study sought to determine if an anti-fatigue mat (AFM) could uniquely affect low back pain (LBP), low back posture, and foot-floor interface responses in individuals prone to developing LBP (termed pain developers (PDs)) during prolonged standing experiments compared to those who do not develop LBP under the same exposures (termed non pain developers (NPDs)). Sixteen volunteers (8 PDs and 8 NPDs) were recruited based on their pain-development tendencies, which were established in previous standing experiments. They visited the laboratory on two separate days for 60 min of light manual work while standing on either a rigid floor or AFM. All participants were asymptomatic at the beginning of each experimental session. The amount of LBP experienced during the standing exposure, measured via a visual analogue scale, was reduced (p = 0.03) in the PD group when on the AFM (3.6 ± 6 mm) compared to the rigid floor (6.8 ± 7 mm). LBP levels remained low and unchanged (p = 0.5) between the AFM (2.4 ± 5 mm) and rigid floor (1.6 ± 2 mm) conditions for the NPD group. Neither postural nor foot-floor interface measures correlated with this unique reduction of LBP for the PD group when standing on the AFM. The AFM did, however, increase centre of pressure excursion (NPD 55% increase; PD 35% increase) and tended to increase the number of body weight shifts (NPD 116% increase; PD 54% increase) in both the PD and NPD groups. These findings suggest that AFMs may selectively benefit individuals prone to developing standing-induced back pain by facilitating subtle movements at the foot-floor interface.

Structural basis for APE1 processing DNA damage in the nucleosome.

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1.

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.

Application of Principal Component Analysis to Forward Reactive Stepping: Whole-body Movement Strategy Differs as a Function of Age and Sex.

BACKGROUND: Differences in reactive stepping strategy to recover balance have been investigated as a function of age and sex, but to date have been measured using discrete step or joint specific measures. It is unknown how whole-body strategy or underlying motor control objectives differ between age and sex groups in forward reactive stepping. RESEARCH QUESTION: Does whole-body movement and/or motor control strategy differ as a function of age or sex in a forward reactive step to maintain balance? METHODS: Forty young and older adults (45 females, 35 males) participated in this study. All participants performed five reactive stepping trials in response to a forward balance perturbation while whole-body kinematics and ground reaction forces were collected. Features of whole-body movement strategy were determined using a principal component analysis model. Average principal component (PC) scores were compared between groups as a measure of whole-body movement strategy and within participant relative standard deviation of PC scores were compared to determine if motor control objectives differed across groups. RESULTS: Significant differences in reactive stepping strategy were observed both as a function of age and sex. Older adults had a greater step length and width, greater anterior trunk and pelvis translation, greater knee flexion angles and anterior translation of the hip joint on the stepping leg compared to young participants. Males had lesser step length and width, as well as greater trunk flexion compared to females. No differences in relative standard deviation of PC scores were observed between age or sex-based groups suggesting that motor control objectives were similar between groups. SIGNIFICANCE: This study demonstrates how whole-body movement strategy differs as a function of age and sex, which explains why previously reported discrete outcomes occur. Additionally, it does not seem that motor control strategy objectives differ between age or sex groups in forward reactive stepping.

Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

The influence of increased passive stiffness of the trunk and hips on balance control during reactive stepping.

BACKGROUND: Age-related changes, which include increased trunk and hip stiffness, negatively influence postural balance. While previous studies suggest no net-effect of trunk and hip stiffness on initial trip-recovery responses, no study to date has examined potential effects during the dynamic restabilisation phase following foot contact. RESEARCH QUESTION: Does increased trunk and hip stiffness, in isolation from other ageing effects, negatively influence balance during the restabilisation phase of reactive stepping. METHODS: Balance perturbations were applied using a tether-release paradigm, which required participants to react with a single-forward step. Sixteen young adults completed two blocks of testing: a baseline and an increased stiffness (corset) condition. Whole-body kinematics were utilized to estimate spatial step parameters, center of mass (COM), COM incongruity (peak - final position) and time to restabilisation, in anteroposterior (AP) and mediolateral (ML) directions. RESULTS: In the corset condition, peak COM displacement was increased in both directions (p < 0.024), which drove reductions in minimum margins of stability (p < 0.032) as step width and length were unchanged (p > 0.233). Increased passive stiffness also increased the magnitude and variability of peak shear ground reaction force, COM incongruity, and time to restabilisation in the ML (but not AP) direction (p < 0.027). SIGNIFICANCE: In contrast to previous literature, increased stiffness resulted in greater peak COM displacement in both directions. Our results suggest increased trunk and hip stiffness have detrimental effects on dynamic stability following a reactive step, particularly in the ML direction. Observed increases in magnitude and variability of COM incongruity suggest the likelihood of a sufficiently large loss of ML stability - requiring additional steps - was increased by stiffening of the hips and trunk. The current findings suggest interventions aiming to mobilize the trunk and hips, in conjunction with strengthening, could improve balance and reduce the risk of falls.