Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein.

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Mechanism of single-stranded DNA annealing by RAD52-RPA complex.

RAD52 is important for the repair of DNA double-stranded breaks1,2, mitotic DNA synthesis3-5 and alternative telomere length maintenance6,7. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA)8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair10. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal11,12, and aberrant expression of RAD52 is associated with poor cancer prognosis13,14. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers15-17. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously18-20, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.

POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor.

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers1-10 and the cancer-prone syndrome Fanconi anaemia11-13. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis.

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2.

DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

The SMX DNA Repair Tri-nuclease.

The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context.

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM­TopIIIα­RMI1­RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

Head Contact and Suspected Concussion Rates in Youth Basketball: Time to Target Head Contact Penalties for Prevention.

OBJECTIVE: To compare game events, head contact (HC) rates, and suspected concussion incidence rates (IRs) in boys' and girls' youth basketball. DESIGN: Cross-sectional. SETTING: Canadian club basketball teams (U16-U18). PARTICIPANTS: Players from 24 boys' and 24 girls' Canadian club basketball teams during the 2022 season. ASSESSMENT OF RISK FACTORS: Recorded games were analyzed using Dartfish video analysis software to compare sexes. MAIN OUTCOME MEASURES: Poisson regression analyses were used to estimate HCs [direct (HC1) and indirect (HC2)], suspected concussion IRs, and IR ratios (IRRs). Game event, court location, and HC1 fouls were reported. RESULTS: Division 1 HC rates did not differ between boys (n = 238; IR = 0.50/10 player-minutes; 95% confidence interval [CI], 0.43-0.56) and girls (n = 220; IR = 0.46/10 player-minutes; 95% CI, 0.40-0.52). Division 2 boys experienced 252 HCs (IR = 0.53/10 player-minutes; 95% CI, 0.46-0.59); girls experienced 192 HCs (IR = 0.40/10 player-minutes; 95% CI, 0.35-0.46). Division 2 boys sustained higher HC1 IRs compared with Division 2 girls (IRR = 1.42; 95% CI, 1.15-1.74). Head contacts, rates did not differ between boys and girls in either Division. Suspected concussion IRs were not significantly different for boys and girls in each Division. Head contacts occurred mostly in the key for boys and girls in each Division. Despite illegality, HC1 penalization ranged from 3.9% to 19.7%. Head contact mechanisms varied across Divisions and sexes. CONCLUSIONS: Despite current safety measures, both HCs and suspected concussions occur in boys' and girls' basketball. Despite the illegality and potential danger associated with HC, only a small proportion of direct HCs were penalized and therefore targeting greater enforcement of these contacts may be a promising prevention target.

Shoulder Check: Investigating Shoulder Injury Rates, Types, Severity, Mechanisms, and Risk Factors in Canadian Youth Ice Hockey.

OBJECTIVE: To describe shoulder-related injury rates (IRs), types, severity, mechanisms, and risk factors in youth ice hockey players during games and practices. DESIGN: Secondary analysis of data from a 5-year prospective cohort study, Safe-to-Play (2013-2018). SETTING: Canadian youth ice hockey. PARTICIPANTS: Overall, 6584 player-seasons (representing 4417 individual players) participated. During this period, 118 shoulder-related games and 12 practice injuries were reported. ASSESSMENT OF RISK FACTORS: An exploratory multivariable mixed-effects Poisson regression model examined the risk factors of body checking policy, weight, biological sex, history of injury in the past 12 months, and level of play. MAIN OUTCOME MEASURES: Injury surveillance data were collected from 2013 to 2018. Injury rates with 95% confidence interval (CI) were estimated using Poisson regression. RESULTS: The shoulder IR was 0.35 injuries/1000 game-hours (95% CI, 0.24-0.49). Two-thirds of game injuries (n = 80, 70%) resulted in >8 days of time-loss, and more than one-third (n = 44, 39%) resulted in >28 days of time-loss. An 83% lower rate of shoulder injury was associated with policy prohibiting body checking compared with leagues allowing body checking (incidence rate ratio [IRR], 0.17; 95% CI, 0.09-0.33). A higher shoulder IR was observed for those who reported any injury in the last 12-months compared with those with no history (IRR, 2.00; 95% CI, 1.33-3.01). CONCLUSIONS: Most shoulder injuries resulted in more than 1 week of time-loss. Risk factors for shoulder injury included participation in a body-checking league and recent history of injury. Further study of prevention strategies specific to the shoulder may merit further consideration in ice hockey.

Parametric g-formula for Testing Time-Varying Causal Effects: What It Is, Why It Matters, and How to Implement It in Lavaan.

Psychologists leverage longitudinal designs to examine the causal effects of a focal predictor (i.e., treatment or exposure) over time. But causal inference of naturally observed time-varying treatments is complicated by treatment-dependent confounding in which earlier treatments affect confounders of later treatments. In this tutorial article, we introduce psychologists to an established solution to this problem from the causal inference literature: the parametric g-computation formula. We explain why the g-formula is effective at handling treatment-dependent confounding. We demonstrate that the parametric g-formula is conceptually intuitive, easy to implement, and well-suited for psychological research. We first clarify that the parametric g-formula essentially utilizes a series of statistical models to estimate the joint distribution of all post-treatment variables. These statistical models can be readily specified as standard multiple linear regression functions. We leverage this insight to implement the parametric g-formula using lavaan, a widely adopted R package for structural equation modeling. Moreover, we describe how the parametric g-formula may be used to estimate a marginal structural model whose causal parameters parsimoniously encode time-varying treatment effects. We hope this accessible introduction to the parametric g-formula will equip psychologists with an analytic tool to address their causal inquiries using longitudinal data.

Coordinated roles of SLX4 and MutSß in DNA repair and the maintenance of genome stability.

SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSß, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSß promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSß with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSß and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSß and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

RTEL1 maintains genomic stability by suppressing homologous recombination.

Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.

Differences in injury and concussion rates in a cohort of Canadian female and male youth Rugby Union: a step towards targeted prevention strategies.

OBJECTIVE: To examine differences in match and training musculoskeletal injury and concussion rates and describe mechanisms of concussion while considering previous playing experience in female and male Canadian high school Rugby Union ('rugby') players. METHODS: A 2-year prospective cohort study was completed in a high school league (n=361 females, 421 player-seasons; n=429 males, 481 player-seasons) in Calgary, Canada over the 2018 and 2019 rugby playing seasons. Baseline testing was completed at the start of each season and injury surveillance and individual player participation through session attendance was documented to quantify individual-level player exposure hours. Injury incidence rates (IRs) and incidence rate ratios (IRRs) were calculated using Poisson regression, offset by player exposure hours and clustered by team. RESULTS: Overall match IR for females was 62% higher than males (overall IRR=1.62, 95% CI: 1.20 to 2.18) and the overall training IR was twice as high for females (overall IRR=2.15, 95% CI: 1.40 to 3.32). The female match concussion IR was 70% higher than the males (concussion IRR=1.70, 95% CI: 1.08 to 2.69). Females had a 75% greater tackle-related IR compared with males (IRR=1.75, 95% CI: 1.20 to 2.56). Additionally, female tacklers had a twofold greater rate of injury compared with male tacklers (IRR=2.17, 95% CI: 1.14 to 4.14). Previous playing experience was not associated with tackle-related injury or concussion IRs. CONCLUSION: The rate of injury and concussion was significantly higher in females within this Canadian high school cohort. These results emphasise the need for development, implementation and evaluation of female-specific injury and concussion prevention strategies to reduce injury and concussion in female youth rugby.

Prevention strategies and modifiable risk factors for sport-related concussions and head impacts: a systematic review and meta-analysis.

OBJECTIVES: To evaluate prevention strategies, their unintended consequences and modifiable risk factors for sport-related concussion (SRC) and/or head impact risk. DESIGN: This systematic review and meta-analysis was registered on PROSPERO (CRD42019152982) and conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. DATA SOURCES: Eight databases (MEDLINE, CINAHL, APA PsycINFO, Cochrane (Systematic Review and Controlled Trails Registry), SPORTDiscus, EMBASE, ERIC0 were searched in October 2019 and updated in March 2022, and references searched from any identified systematic review. ELIGIBILITY CRITERIA: Study inclusion criteria were as follows: (1) original data human research studies, (2) investigated SRC or head impacts, (3) evaluated an SRC prevention intervention, unintended consequence or modifiable risk factor, (4) participants competing in any sport, (5) analytic study design, (6) systematic reviews and meta-analyses were included to identify original data manuscripts in reference search and (7) peer-reviewed. Exclusion criteria were as follows: (1) review articles, pre-experimental, ecological, case series or case studies and (2) not written in English. RESULTS: In total, 220 studies were eligible for inclusion and 192 studies were included in the results based on methodological criteria as assessed through the Scottish Intercollegiate Guidelines Network high ('++') or acceptable ('+') quality. Evidence was available examining protective gear (eg, helmets, headgear, mouthguards) (n=39), policy and rule changes (n=38), training strategies (n=34), SRC management strategies (n=12), unintended consequences (n=5) and modifiable risk factors (n=64). Meta-analyses demonstrated a protective effect of mouthguards in collision sports (incidence rate ratio, IRR 0.74; 95% CI 0.64 to 0.89). Policy disallowing bodychecking in child and adolescent ice hockey was associated with a 58% lower concussion rate compared with bodychecking leagues (IRR 0.42; 95% CI 0.33 to 0.53), and evidence supports no unintended injury consequences of policy disallowing bodychecking. In American football, strategies limiting contact in practices were associated with a 64% lower practice-related concussion rate (IRR 0.36; 95% CI 0.16 to 0.80). Some evidence also supports up to 60% lower concussion rates with implementation of a neuromuscular training warm-up programme in rugby. More research examining potentially modifiable risk factors (eg, neck strength, optimal tackle technique) are needed to inform concussion prevention strategies. CONCLUSIONS: Policy and rule modifications, personal protective equipment, and neuromuscular training strategies may help to prevent SRC. PROSPERO REGISTRATION NUMBER: CRD42019152982.