Biology subjects

Smith, D. J.

Publications and source records attributed to Smith, D. J..

54 records · Page 3Linked to original sources

bioRxiv2019

Rapid sperm capture: High-throughput flagellar waveform analysis

Flagella are critical across all eukaryotic life, and the human sperm flagellum is crucial to natural fertility. Existing automated sperm diagnostics (CASA) rely on tracking the sperm head and extrapolating measures. We describe fully-automated tracking and analysis of flagellar movement for large cell numbers. The analysis is demonstrated on freely-motile cells in low and high viscosity fluids, and validated on published data of tethered cells undergoing pharmacological hyperactivation. Direct analysis of the flagellar beat reveals that the CASA measure beat cross frequency, does not measure beat frequency. A new measurement, track centroid speed, is validated as an accurate differentiator of progressive motility. Coupled with fluid mechanics codes, waveform data enables extraction of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses. We provide a powerful and accessible research tool, enabling connection of the cells mechanical activity to its motility and effect on its environment.

cell biology↗

bioRxiv2019

The genomic basis of mood instability: identification of 46 loci in 363,705 UK Biobank participants, genetic correlation with psychiatric disorders, and association with gene expression and function.

Genome-wide association studies (GWAS) of psychiatric phenotypes have tended to focus on categorical diagnoses, but to understand the biology of mental illness it may be more useful to study traits which cut across traditional boundaries. Here we report the results of a GWAS of mood instability (MI) as a trait in a large population cohort (UK Biobank, n=363,705). We also assess the clinical and biological relevance of the findings, including whether genetic associations show enrichment for nervous system pathways. Forty six unique loci associated with MI were identified with a heritability estimate of 9%. Linkage Disequilibrium Score Regression (LDSR) analyses identified genetic correlations with Major Depressive Disorder (MDD), Bipolar Disorder (BD), Schizophrenia (SZ), anxiety and Post Traumatic Stress Disorder (PTSD). Gene-level and gene set analyses identified total 244 significant genes and 6 enriched gene sets. Tissue expression analysis from the SNP level data found enrichment in multiple brain regions, and eQTL analyses highlighted an inversion on chromosome 17 plus two brain-specific eQTLs. Additionally, we used a Phenotype Linkage Network (PLN) analysis and community analysis to assess for enrichment of nervous system gene sets using mouse orthologue databases. The PLN analysis found enrichment in nervous system PLNs for a community containing serotonin and melatonin receptors. In summary, this work has identified novel loci, tissues, and gene sets contributing to MI as a normal trait and will inform future work on the biology of mood and psychotic disorders, and to point the way towards potential for new stratified medicine approaches and the identification of novel trans-diagnostic drug targets.

genetics↗

bioRxiv2019

Stratifying depression by neuroticism: revisiting a diagnostic tradition using GWAS data

Major depressive disorder and neuroticism share a large genetic basis. We sought to determine whether this shared basis could be decomposed to identify genetic factors that are specific to depression. We analysed two sets of summary statistics from genome-wide association studies of depression (from the Psychiatric Genomics Consortium and 23andMe) and compared them to GWAS of neuroticism (from UK Biobank). First, we used a pairwise GWAS analysis to classify variants as associated with only depression, with only neuroticism, or with both. Second, we estimated partial genetic correlations to test whether the depressions genetic link with other phenotypes was explained by shared overlap with neuroticism. We found evidence that most genetic variants associated with depression are likely to be shared with neuroticism. The overlapping common genetic variance of depression and neuroticism was negatively genetically correlated with cognitive function and positively genetically correlated with several psychiatric disorders. We found that the genetic contributions unique to depression, and not shared with neuroticism, were correlated with inflammation, cardiovascular disease, and sleep patterns. Our findings demonstrate that, while genetic risk factors for depression are largely shared with neuroticism, there are also non-neuroticism related features of depression that may be useful for further patient or phenotypic stratification.

genetics↗

bioRxiv2018

Epistatic interactions can moderate the antigenic effect of substitutions in hemagglutinin of influenza H3N2 virus

We previously showed that single amino acid substitutions at seven positions in hemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in eleven representative H3 hemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at hemagglutinin position 145 was fully independent of the amino acid context of the representative hemagglutinins. Antigenic change caused by substitutions introduced at hemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the hemagglutinin can moderate the magnitude of antigenic change.

microbiology↗

bioRxiv2018

Identification of novel common variants associated with chronic pain using conditional false discovery rate analysis with major depressive disorder and assessment of pleiotropic effects of LRFN5.

Chronic pain is highly prevalent worldwide, with a significant socioeconomic burden, and also contributes to excess mortality. Chronic pain is a complex trait that is moderately heritable and genetically, as well as phenotypically, correlated with major depressive disorder (MDD). Use of the Conditional False Discovery Rate (cFDR) approach, which leverages pleiotropy identified from existing GWAS outputs, has been successful in discovering novel associated variants in related phenotypes. Here, genome-wide association study outputs for both von Korff chronic pain grade as a quasi-quantitative trait and for MDD were used to identify variants meeting a cFDR threshold for each outcome phenotype separately, as well as a conjunctional cFDR (ccFDR) threshold for both phenotypes together. Using a moderately conservative threshold, we identified a total of 11 novel single nucleotide polymorphisms (SNPs), six of which were associated with chronic pain grade and nine of which were associated with MDD. Four SNPs on chromosome 14 were associated with both chronic pain grade and MDD. SNPs associated only with chronic pain grade were located within SLC16A7 on chromosome 12. SNPs associated only with MDD were located either in a gene-dense region on chromosome 1 harbouring LINC01360, LRRIQ3, FPGT and FPGT-TNNI3K, or within/close to LRFN5 on chromosome 14. The SNPs associated with both outcomes were also located within LRFN5. Several of the SNPs on chromosomes 1 and 14 were identified as being associated with expression levels of nearby genes in the brain and central nervous system. Overall, using the cFDR approach, we identified several novel genetic loci associated with chronic pain and we describe likely pleiotropic effects of a recently identified MDD locus on chronic pain.\n\nAuthor SummaryGenetic variants explaining variation in complex traits can often be associated with more than one trait at once ( pleiotropy). Taking account of this pleiotropy in genetic studies can increase power to find sites in the genome harbouring trait-associated variants. In this study we used the suspected underlying pleiotropy between chronic pain and major depressive disorder to discover novel variants associated with chronic pain, and to investigate genetic variation that may be shared between the two disorders.

genetics↗

bioRxiv2018

Genome-wide Association Study of Multisite Chronic Pain in UK Biobank

Chronic pain is highly prevalent worldwide, contributing a significant socioeconomic and public health burden. Several aspects of chronic pain, for example back pain and a severity-related phenotype, chronic pain grade, have been shown to be complex, heritable traits with a polygenic component. Additional pain-related phenotypes capturing aspects of an individuals overall sensitivity to experiencing and reporting chronic pain have also been suggested. We have here made use of a measure of the number of sites of chronic pain in individuals within the general UK population. This measure, termed Multisite Chronic Pain (MCP), is also a complex trait, but its genetic architecture has not previously been investigated. To address this, a large-scale genome-wide association study (GWAS) of MCP was carried out in ~380,000 UK Biobank participants to identify associated genetic variants. Findings were consistent with MCP having a significant polygenic component with a SNP heritability of 10.2%, and 76 independent lead single nucleotide polymorphisms (SNPs) at 39 risk loci were identified. Additional gene-level association analyses identified neurogenesis, synaptic plasticity, nervous system development, cell-cycle progression and apoptosis genes as being enriched for genetic association with MCP. Genetic correlations were observed between MCP and a range of psychiatric, autoimmune and anthropometric traits including major depressive disorder (MDD), asthma and BMI. Furthermore, in Mendelian randomisation (MR) analyses a bi-directional causal relationship was observed between MCP and MDD. A polygenic risk score (PRS) for MCP was found to significantly predict chronic widespread pain (pain all over the body), indicating the existence of genetic variants contributing to both of these pain phenotypes. These findings support the proposition that chronic pain involves a strong nervous system component and have implications for our understanding of the physiology of chronic pain and for the development of novel treatment strategies.

genetics↗

bioRxiv2018

Genome-wide meta-analysis of depression in 807,553 individuals identifies 102 independent variants with replication in a further 1,507,153 individuals

Major depression is a debilitating psychiatric illness that is typically associated with low mood, anhedonia and a range of comorbidities. Depression has a heritable component that has remained difficult to elucidate with current sample sizes due to the polygenic nature of the disorder. To maximise sample size, we meta-analysed data on 807,553 individuals (246,363 cases and 561,190 controls) from the three largest genome-wide association studies of depression. We identified 102 independent variants, 269 genes, and 15 gene-sets associated with depression, including both genes and gene-pathways associated with synaptic structure and neurotransmission. Further evidence of the importance of prefrontal brain regions in depression was provided by an enrichment analysis. In an independent replication sample of 1,306,354 individuals (414,055 cases and 892,299 controls), 87 of the 102 associated variants were significant following multiple testing correction. Based on the putative genes associated with depression this work also highlights several potential drug repositioning opportunities. These findings advance our understanding of the complex genetic architecture of depression and provide several future avenues for understanding aetiology and developing new treatment approaches.

genetics↗

bioRxiv2018

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5 flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5 flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

molecular biology↗

bioRxiv2018

Genome-wide association study of circadian rhythmicity in 71 500 UK Biobank participants and polygenic association with mood instability

BackgroundCircadian rhythms are fundamental to health and are particularly important for mental wellbeing. Disrupted rhythms of rest and activity are recognised as risk factors for major depressive disorder and bipolar disorder.\n\nMethodsWe conducted a genome-wide association study (GWAS) of low relative amplitude (RA), an objective measure of circadian rhythmicity derived from the accelerometer data of 71 500 UK Biobank participants. Polygenic risk scores (PRS) for low RA were used to investigate potential associations with psychiatric phenotypes.\n\nOutcomesTwo independent genetic loci were associated with low RA, within genomic regions for Neurofascin (NFASC) and Solute Carrier Family 25 Member 17 (SLC25A17). A secondary GWAS of RA as a continuous measure identified a locus within Meis Homeobox 1 (MEIS1). There were no significant genetic correlations between low RA and any of the psychiatric phenotypes assessed. However, PRS for low RA was significantly associated with mood instability across multiple PRS thresholds (at PRS threshold 0{middle dot}05: OR=1{middle dot}02, 95% CI=1{middle dot}01-1{middle dot}02, p=9{middle dot}6x10-5), and with major depressive disorder (at PRS threshold 0{middle dot}1: OR=1{middle dot}03, 95% CI=1{middle dot}01-1{middle dot}05, p=0{middle dot}025) and neuroticism (at PRS threshold 0{middle dot}5: Beta=0{middle dot}02, 95% CI=0{middle dot}007-0{middle dot}04, p=0{middle dot}021).\n\nInterpretationOverall, our findings contribute new knowledge on the complex genetic architecture of circadian rhythmicity and suggest a putative biological link between disrupted circadian function and mood disorder phenotypes, particularly mood instability, but also major depressive disorder and neuroticism.

genomics↗

bioRxiv2018

Separable recruitment of DNA Polymerase α for leading- and lagging-strand replication initiation

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol ) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol abundance, and that both processes are disrupted at similar Pol concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol , lagging-strand initiation is impaired at Pol concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol . Using strains in which the Pol -Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol at the replication fork, at least when Pol is limiting. Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases {varepsilon} and {delta} on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase -primase (Pol ). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

molecular biology↗

bioRxiv2018

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

molecular biology↗

bioRxiv2018

Polygenic risk scores for major depressive disorder and neuroticism as predictors of antidepressant response: meta-analysis of three treatment cohorts.

There are currently no reliable approaches for correctly identifying which patients with major depressive disorder (MDD) will respond well to antidepressant therapy. However, recent genetic advances suggest that Polygenic Risk Scores (PRS) could allow MDD patients to be stratified for antidepressant response. We used PRS for MDD and PRS for neuroticism as putative predictors of antidepressant response within three treatment cohorts: The Genome-based Therapeutic Drugs for Depression (GENDEP) cohort, and 2 sub-cohorts from the Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomics Study PRGN-AMPS (total patient number = 783). Results across cohorts were combined via meta-analysis within a random effects model. Overall, PRS for MDD and neuroticism did not significantly predict antidepressant response but there was a consistent direction of effect, whereby greater genetic loading for both MDD (best MDD result, p < 5*10-5 MDD-PRS at 4 weeks, {beta} = -0.019, S.E = 0.008, p = 0.01) and neuroticism (best neuroticism result, p < 0.1 neuroticism-PRS at 8 weeks, {beta} = -0.017, S.E = 0.008, p = 0.03) were associated with less favourable response. We conclude that the PRS approach may offer some promise for treatment stratification in MDD and should now be assessed within larger clinical cohorts.

genetics↗

bioRxiv2017

Is there association between APOE e4 genotype and structural brain ageing phenotypes, and does that association increase in older age in UK Biobank? (N = 8,395)

Apolipoprotein (APOE) e4 genotype is a purported risk factor for accelerated cognitive ageing and dementia, though its neurostructural substrates are unclear. The deleterious effects of this genotype on brain structure may increase in magnitude into older age. This study aimed to investigate in UK Biobank the association between APOE e4 allele presence vs. absence and brain imaging variables that have been associated with worse cognitive abilities; and whether this association varies by cross-sectional age. We used brain magnetic resonance imaging (MRI) and genetic data from a general-population cohort: the UK Biobank (N=8,395). We adjusted for the covariates of age in years, sex, Townsend social deprivation scores, smoking history and cardiometabolic diseases. There was a statistically significant association between APOE e4 genotype and increased (i.e. worse) white matter (WM) hyperintensity volumes (standardised beta = 0.088, 95 confidence intervals = 0.036 to 0.139, P = 0.001), a marker of poorer cerebrovascular health. There were no associations with left or right hippocampal, total grey matter (GM) or WM volumes, or WM tract integrity indexed by fractional anisotropy (FA) and mean diffusivity (MD). There were no statistically significant interactions with age. Future research in UK Biobank utilising intermediate phenotypes and longitudinal imaging hold significant promise for this area, particularly pertaining to APOE e4s potential link with cerebrovascular contributions to cognitive ageing.

genetics↗

bioRxiv2017

Genome-wide analysis of risk-taking behaviour and cross-disorder genetic correlations in 116,255 individuals from the UK Biobank cohort

Risk-taking behaviour is a key component of several psychiatric disorders and could influence lifestyle choices such as smoking, alcohol use and diet. As a phenotype, risk-taking behaviour therefore fits within a Research Domain Criteria (RDoC) approach, whereby identifying genetic determinants of this trait has the potential to improve our understanding across different psychiatric disorders. Here we report a genome wide association study in 116 255 UK Biobank participants who responded yes/no to the question \"Would you consider yourself a risk-taker?\" Risk-takers (compared to controls) were more likely to be men, smokers and have a history of psychiatric disorder. Genetic loci associated with risk-taking behaviour were identified on chromosomes 3 (rs13084531) and 6 (rs9379971). The effects of both lead SNPs were comparable between men and women. The chromosome 3 locus highlights CADM2, previously implicated in cognitive and executive functions, but the chromosome 6 locus is challenging to interpret due to the complexity of the HLA region. Risk-taking behaviour shared significant genetic risk with schizophrenia, bipolar disorder, attention deficit hyperactivity disorder and post-traumatic stress disorder, as well as with smoking and total obesity. Despite being based on only a single question, this study furthers our understanding of the biology of risk-taking behaviour, a trait which has a major impact on a range of common physical and mental health disorders.

genetics↗

bioRxiv2017

Genome-wide association study of depression phenotypes in UK Biobank (n = 322,580) identifies the enrichment of variants in excitatory synaptic pathways

Depression is a polygenic trait that causes extensive periods of disability and increases the risk of suicide, a leading cause of death in young people. Previous genetic studies have identified a number of common risk variants which have increased in number in line with increasing sample sizes. We conducted a genome-wide association study (GWAS) in the largest single population-based cohort to date, UK Biobank. This allowed us to estimate the effects of {approx} 8 million genetic variants in 320,000 people for three depression phenotypes: broad depression, probable major depressive disorder (MDD), and International Classification of Diseases (ICD, version 9 or 10)-coded MDD. Each phenotype was found to be significantly genetically correlated with the results from a previous independent study of clinically defined MDD. We identified 14 independent loci that were significantly associated (P < 5 x 10-8) with broad depression, two independent variants for probable MDD, and one independent variant for ICD-coded MDD. Gene-based analysis of our GWAS results with MAGMA revealed 46 regions significantly associated (P < 2.77 x 10-6) with broad depression, two significant regions for probable MDD and one significant region for ICD-coded MDD. Gene region-based analysis of our GWAS results with MAGMA revealed 59 regions significantly associated (P < 6.02 x 10-6) with broad depression, of which 27 were also detected by gene-based analysis. Variants for broad depression were enriched in pathways for excitatory neurotransmission, mechanosensory behavior, postsynapse, neuron spine and dendrite. This study provides a number of novel genetic risk variants that can be leveraged to elucidate the mechanisms of MDD and low mood.

genetics↗

bioRxiv2017

Genome-Wide Analysis Of 113,968 Individuals In UK Biobank Identifies Four Loci Associated With Mood Instability.

Mood instability is a core clinical feature of affective and psychotic disorders. In keeping with the Research Domain Criteria (RDoC) approach, it may be a useful construct for identifying biology that cuts across psychiatric categories. We aimed to investigate the biological validity of a simple measure of mood instability and evaluate its genetic relationship with several psychiatric disorders, including major depressive disorder (MDD), bipolar disorder (BD), schizophrenia, attention deficit hyperactivity disorder (ADHD), anxiety disorder and post-traumatic stress disorder (PTSD). We conducted a genome-wide association study (GWAS) of mood instability in 53,525 cases and 60,443 controls from UK Biobank, identifying four independently-associated loci (on chromosomes eight, nine, 14 and 18), and a common single nucleotide polymorphism (SNP)-based heritability estimate of approximately 8%. We found a strong genetic correlation between mood instability and MDD (rg=0.60, SE=0.07, p=8.95 x 10-17) and a small but significant genetic correlation with both schizophrenia (rg=0.11, SE=0.04, p=0.01) and anxiety disorders (rg=0.28, SE=0.14, p=0.04), although no genetic correlation with BD, ADHD or PTSD. Several genes at the associated loci may have a role in mood instability, including the DCC netrin 1 receptor (DCC) gene, eukaryotic translation initiation factor 2B subunit beta (eIF2B2), placental growth factor (PGF), and protein tyrosine phosphatase, receptor type D (PTPRD). Strengths of this study include the very large sample size, but our measure of mood instability may be limited by the use of a single question. Overall, this work suggests a polygenic basis for mood instability. This simple measure can be obtained in very large samples; our findings suggest that doing so may offer the opportunity to illuminate the fundamental biology of mood regulation.

neuroscience↗

bioRxiv2016

Cannabis use and risk of schizophrenia:a Mendelian randomization study

Cannabis use is observationally associated with an increased risk of schizophrenia, however whether the relationship is causal is not known. To determine the nature of the association between cannabis use on risk of schizophrenia using Mendelian randomization (MR) analysis, we used ten genetic variants previously identified to associate with cannabis use in 32,330 individuals. Genetic variants were used in a MR analyses of the association of genetically determined cannabis on risk of schizophrenia in 34,241 cases and 45,604 controls from predominantly European descent. Estimates from MR were compared to a metaanalysis of observational studies reporting effect estimates for ever use of cannabis and risk of schizophrenia or related disorders. Genetically determined use of cannabis was associated with increased risk of schizophrenia (OR of schizophrenia for users vs. non-users of cannabis: 1.37; 95%CI, 1.09 to 1.67; P-value=0.007). The corresponding estimate from observational analysis was 1.50 (95% CI, 1.10 to 2.00; P-value for heterogeneity = 0.88). The genetic instrument did not show evidence of pleiotropy on MR-Egger (Egger test, P-value=0.292) nor on multivariable MR accounting for tobacco exposure (OR of schizophrenia for users vs. nonusers of cannabis, adjusted for ever vs. never smoker: 1.41; 95% CI, 1.09-1.83). Furthermore, the causal estimate remained robust to sensitivity analyses. These findings strongly support a causal association between genetically determined use of cannabis and risk of schizophrenia. Such robust evidence may inform public health message about the risks of cannabis use, especially regarding its potential mental health consequences.

epidemiology↗

bioRxiv2016

Pif1-family helicases cooperate to suppress widespread replication fork arrest at tRNA genes

Saccharomyces cerevisiae encodes two distinct Pif1-family helicases - Pif1 and Rrm3 - which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase {delta} during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in the S. cerevisiae genome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.

molecular biology↗