Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA.

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA-origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres-suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

Pediatric Firearm-Related Injuries: Taking Kids Out of Harm's Way Begins with Targeted Prevention.

BACKGROUND: Firearm-related injury is a public health crisis and remains the 3rd most common cause of death from ages 1 15 years. By evaluating events surrounding such injuries, evidence-based intervention strategies efforts may be targeted to maximize impact. MATERIAL AND METHODS: A retrospective chart review was performed for firearm-related injuries in patients 15 years-old and under at an urban Pediatric Level 1 Trauma Center between January 2016 and December 2020. Age, gender, race/ethnicity, injury severity score (ISS), reported cause of injury, timing of injury around school and curfew, and mortality were evaluated. Medical Examiner data identified other deaths that occurred within the hospital's catchment area. RESULTS: There were 195 injuries including 14 from the Medical Examiner. Overall, 82.6% were male with median age of 14 years (range 1-15; IQR 13-15), and median ISS of 5 (IQR 1-10). African-American children comprised 74.9% of the cohort while only representing 35.9% of local schools. Intentional interpersonal injuries comprised 65.6%; 17.4% were bystanders; 7.2% were negligent discharges; and 0.5% suicide. Median age for intentional interpersonal injuries was 14 years (IQR 13-15) compared to 11 years (IQR 8-14, P = 0.03) for negligent discharges. Regarding timing, 6.9% of injuries occurred during school hours; 56.4% after school or during non-school days; and 36.7% were after legal curfew. Mortality rate was 17.4%. CONCLUSIONS: Firearm safety education and community-based violence intervention should start in the pre-teen years. Pediatric firearm-related injury prevention strategies must be multifaceted addressing structural racism, truancy, curfew violation, extra-curricular activities, childcare options, firearm safety education, violence reduction, suicide prevention, and recidivism.

Early venoarterial extracorporeal membrane oxygenation improves outcomes in post-cardiotomy shock.

Post-cardiotomy shock (PCS) is associated with substantial morbidity and mortality. We reviewed our 12-year experience of venoarterial extracorporeal membrane oxygenation (VA-ECMO) therapy for PCS. Between July 2007 and June 2018, 156 consecutive patients underwent VA-ECMO for PCS. We retrospectively investigated patient characteristics, indications, and management to determine factors affecting outcomes. Secondary analysis was performed by dividing the cohort into Era 1 (2007-2012, n = 52) and Era 2 (2013-2018, n = 104) for comparison. After a median of 4.70 days (interquartile range [IQR] 2.76-8.53) of ECMO support, 72 patients (46.1%) survived to discharge. In-hospital mortality decreased in Era 2 from 75 to 43.3% (P < 0.001). Survivors were cannulated at lower serum lactate (5.3 [IQR 2.8-8.2] versus 7.5 [4.7-10.7], P = 0.003) and vasoactive-inotropic score (22.7 [IQR 11.3-35.5] versus 28.1 [IQR 20.8-42.5], P = 0.017). Patients in Era 2 were more frequently cannulated intraoperatively (63.5% versus 34.6%, P = 0.002), earlier in their hospital course, and at lower levels of serum lactate and vasoactive-inotropic score than in Era 1. Independent risk factors for mortality included increased age (odds ratio [OR] 1.06, P = 0.002), serum lactate at cannulation (OR 1.17, P = 0.009), and vasoactive-inotropic score (OR 1.04, P = 0.009). Bleeding and limb ischemia were less common in Era 2. Overall, outcomes of ECMO for PCS improved over the study period. The survival benefit appears to be associated with earlier ECMO initiation before prolonged hypoperfusion occurs.

Considerations for Referral: What Happens to Patients After Being Turned Down for Left Ventricular Assist Device Therapy.

BACKGROUND: Left ventricular assist device (LVAD) therapy has revolutionized the treatment options for patients with advanced heart failure. Patient selection is essential for obtaining successful results. However, few data exist concerning the outcomes of patients evaluated for LVAD therapy but subsequently rejected or deferred. METHODS AND RESULTS: This is a retrospective review of all patients referred for LVAD therapy at our institution between January 2009 and December 2016. Baseline demographics and Interagency Registry for Mechanically Assisted Circulatory Support profiles were collected, and reasons for rejection or deferral for LVAD placement were investigated. A total of 669 patients were referred for LVAD therapy, and 228 patients (34%) were turned down. The yearly acceptance rate ranged between 57% and 75%. The average age of the turned-down cohort was 60.8 ± 12.5 years; 83% were men. Reasons for rejection included: patient being too sick (34%); psychosocial concerns (25%); patient declined (16%); decision was deferred for medical optimization (15%); or patient being too well (10%). The percentage of patients who were rejected due to psychosocial concerns has increased over time (P = 0.02), whereas the rate of deferral for medical optimization has remained stable (P = 0.10). One-year survival after initial LVAD consultation was 42% in those who were too sick, 64% in those with psychosocial concerns, 68% in patients who declined, 86% in those deferred for medical optimization; and 100% in those too well (P < 0.01). CONCLUSIONS: One-year survival is reduced among patients who were initially turned down for LVAD therapy, except for those in whom this decision was deferred for medical optimization or because the patient was too well. Psychosocial concerns have become a significant barrier to LVAD therapy.

A novel in vivo assessment of fluid dynamics on aortic valve leaflet using epi-aortic echocardiogram.

BACKGROUND: Mechanical stress caused by blood flow, such as wall shear stress (WSS) and its related parameters, is key moderator of endothelial degeneration. However, an in vivo method to measure WSS on heart valves has not been developed. METHODS: We developed a novel approach, based on vector flow mapping using intraoperative epi-aortic echocardiogram, to measure WSS and oscillatory shear index (OSI) on the aortic valve. We prospectively enrolled 15 patients with normal valves, who underwent coronary artery bypass graft. RESULTS: Systolic WSS on the ventricularis (2.40 ± 0.44 Pa [1.45-3.00 Pa]) was higher than systolic WSS on the fibrosa (0.33 ± 0.08 Pa [0.14-0.47 Pa], P < .001) and diastolic WSS on the ventricularis (0.18 ± 0.07 Pa [0.04-0.28 Pa], P < .001). Oscillatory shear index on the fibrosa was higher than on the ventricularis (0.29 ± 0.04 [0.24-0.36] vs 0.05 ± 0.03 [0.01-0.12], P < .001). A pilot study involving two patients with severe aortic regurgitation showed significantly different values in fluid dynamics. CONCLUSION: Vector flow mapping method using intraoperative epi-aortic echocardiogram is an effective way of measuring WSS and OSI on normal aortic leaflet in vivo, allowing for better understanding of the pathophysiology of aortic valve diseases.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

Ten-year outcomes of extracorporeal life support for in-hospital cardiac arrest at a tertiary center.

Extracorporeal cardiopulmonary resuscitation (ECPR) is controversial, given both the lack of evidence for improved outcomes and clarity on appropriate candidacy during time-sensitive cardiac arrest situations. The primary objective of our study was to identify factors predicting successful outcomes in ECPR patients.Between March 2007 and November 2018, 112 patients were placed on extracorporeal life support (ECLS) during active CPR (ECPR) at our institution. The primary outcome was survival to hospital discharge. Survivors and non-survivors were compared in terms of pre-cannulation comorbidities, laboratory values, and overall outcomes. Multivariable logistic regression was used to identify pre-cannulation predictors of in-hospital mortality. Among 112 patients, 44 (39%) patients survived to decannulation and 31 (28%) survived to hospital discharge. The median age was 60 years (IQR 45-72) with a median ECLS duration of 2.2 days (IQR 0.6-5.1). Patients who survived to discharge had lower rates of chronic kidney disease than non-survivors (19% vs. 41%, p = 0.046) and lower baseline creatinine values [median 1.2 mg/dL (IQR 0.8-1.7) vs. 1.7 (0.7-2.7), p = 0.008]. Median duration from CPR initiation to cannulation was 40 min (IQR 30-50) with no difference between survivors and non-survivors (p = 0.453). When controlling for age and CPR duration, multivariable logistic regression with pre-procedural risk factors identified pre-arrest serum creatinine as an independent predictor of mortality [OR 3.25 (95% CI 1.22-8.70), p = 0.019] and higher pre-arrest serum albumin as protective [OR 0.32 (95% CI 0.14-0.74), p = 0.007]. In our cohort, pre-arrest creatinine and albumin were independently predictive of in-hospital mortality during ECPR, while age and CPR duration were not.

Outcomes of mechanical support for cardiogenic shock associated with late cardiac allograft failure.

BACKGROUND: Late graft failure (LGF) is an unresolved issue after orthotopic heart transplant (OHT). In this study, we report characteristics and outcomes of severe LGF requiring mechanical circulatory support (MCS). METHODS: All patients undergoing OHT from 2000 to 2018 at our center were reviewed. Patients re-admitted to the hospital for late graft failure (>3 months after initial discharge) and developing cardiogenic shock requiring MCS were identified. Outcomes and mortality were evaluated. RESULTS: Twenty-six patients were identified. Median age was 37.3 years (interquartile range: 28.2-47.6) and 69% were male. Median time from initial transplant to MCS was 2.9 years. Etiology of graft failure was rejection in 19 patients (73%), transplant coronary artery disease (tCAD) in 3 (12%), with mixed tCAD or rejection in 4 (15%).

Sex-related difference in outcomes after aortic root replacement.

PURPOSE: Poorer short-term outcomes have been described for females after cardiovascular surgery. We examined the influence of sex on the outcomes after aortic root replacement (ARR). METHODS: Medical records of 848 patients (females, n = 159/848, 19%) who underwent ARR at our center from 2005 to 2018 were retrospectively reviewed. Sex differences of the following outcomes were analyzed: the primary end point (in-hospital mortality or stro111ke), secondary end point (new requirement for permanent pacemaker), and long-term survival (median follow-up 21.4 months [interquartile range,1.3-60.0]). RESULTS: Females were significantly older (61.3 vs 58.7 [male]) with higher rates of pre-existing cerebrovascular disease (14% [22/159] vs 7% [52/689]) and previous valve intervention (20% [32/159] vs 13% [89/689]) but less myocardial infarction [1%(1/159) vs 7%(48/689)]. The surgical indication was different (aneurysm 75% [120/159] vs 87% [602/689], dissection 13% [21/159] vs 6% [41/689]; P < .01]). Females had larger average aneurysm size after controlling for body size (P ≤ .001). There was no sex difference in in-hospital mortality (3% [5/159] vs 2% [16/689]) or stroke (4% [7/159] vs 4% [29/689]). Multivariable logistic regression indicated that female sex was not an independent predictor of combined in-hospital stroke or death (odds ratio [OR], 0.59; 95% confidence interval [CI], 0.28-1.25), confirmed by propensity score analysis. There was no difference in long-term survival (5-year survival, 90.96% vs 93.03%; P = .44). Females had higher incidence of permanent pacemaker requirement [11% (18/159) vs 6% (39/689), P = .03] and female sex was an independent predictor of permanent pacemaker requirement (OR, 2.01; 95% CI, 1.085-3.724; P = .03). CONCLUSIONS: While female patients have different baseline characteristics and indication for ARR, they are not exposed to an increased risk of in-hospital mortality or stroke. However, females experience increased incidence of permanent pacemaker requirement.

Studying absorbance properties and mercury remediation capabilities of gold-graphene oxide-iron oxide (Au-GO-Fe3O4) nanoparticle systems.

Mercury pollution is a rampant problem in many economically significant Philippine freshwater ecosystems. Communities dependent on these freshwater sources are therefore at risk for exposure to harmful levels of mercury. Various formulations of a novel gold-graphene oxide-iron oxide (Au-GO-Fe3O4) hybrid nanoparticle system were created and subjected to UV-Vis spectroscopy to determine optimal formulations that would best serve as potential substrates for Surface-Enhanced Raman Spectroscopy (SERS) detection of mercury. Optimal formulations of Au-GO-Fe3O4 were also introduced into mercury-polluted environments to evaluate its ability to remove mercury from both water and biological tissues. Spectroscopic analysis revealed that Fe3O4-rich formulations of Au-GO-Fe3O4 had the greatest potential to boost Raman signal intensities of mercury due to red shifting of absorbance peaks and overall increased absorbance across visible wavelengths resulting in the inclusion of greater areas underneath absorbance peaks. Mercury remediation experiments likewise demonstrated Au-GO-Fe3O4 to significantly reduce average concentrations of mercury from 1.67 to 0.82 ppm in polluted water samples - corresponding to a mercury removal efficiency of 50.9% and a mercury adsorption capacity of 5.89 mg/g. The results highlight the viability of Au-GO-Fe3O4 to function as both substrate for SERS detection of mercury and as effective adsorbent for mercury remediation.

The projected timing of climate departure from recent variability.

Ecological and societal disruptions by modern climate change are critically determined by the time frame over which climates shift beyond historical analogues. Here we present a new index of the year when the projected mean climate of a given location moves to a state continuously outside the bounds of historical variability under alternative greenhouse gas emissions scenarios. Using 1860 to 2005 as the historical period, this index has a global mean of 2069 (±18 years s.d.) for near-surface air temperature under an emissions stabilization scenario and 2047 (±14 years s.d.) under a 'business-as-usual' scenario. Unprecedented climates will occur earliest in the tropics and among low-income countries, highlighting the vulnerability of global biodiversity and the limited governmental capacity to respond to the impacts of climate change. Our findings shed light on the urgency of mitigating greenhouse gas emissions if climates potentially harmful to biodiversity and society are to be prevented.

The SWI/SNF ATP-dependent nucleosome remodeler promotes resection initiation at a DNA double-strand break in yeast.

DNA double-strand breaks (DSBs) are repaired by either the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway. Pathway choice is determined by the generation of 3΄ single-strand DNA overhangs at the break that are initiated by the action of the Mre11-Rad50-Xrs2 (MRX) complex to direct repair toward HR. DSB repair occurs in the context of chromatin, and multiple chromatin regulators have been shown to play important roles in the repair process. We have investigated the role of the SWI/SNF ATP-dependent nucleosome-remodeling complex in the repair of a defined DNA DSB. SWI/SNF was previously shown to regulate presynaptic events in HR, but its function in these events is unknown. We find that in the absence of functional SWI/SNF, the initiation of DNA end resection is significantly delayed. The delay in resection initiation is accompanied by impaired recruitment of MRX to the DSB, and other functions of MRX in HR including the recruitment of long-range resection factors and activation of the DNA damage response are also diminished. These phenotypes are correlated with a delay in the eviction of nucleosomes surrounding the DSB. We propose that SWI/SNF orchestrates the recruitment of a pool of MRX that is specifically dedicated to HR.

Identifying New Small Proteins in Escherichia coli.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.

Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

Differences in Telemedicine Use Between People With and Without an Intellectual or Other Developmental Disability During the COVID-19 Pandemic.

Telemedicine utilization of people with an Intellectual or Other Developmental Disability (IDD) during the COVID-19 Pandemic is not well known. This study compares telemedicine utilization of those with and without IDD prior to the pandemic to after it began. Using the Utah All Payers Claims Database from 2019 to 2021, the study identified telemedicine utilization of adults aged 18 to 62 years old in 2019. Propensity score matching was used to minimize observed confounders of subjects with and without IDD in 2019. Negative binomial regression was used to identify factors that were associated with telemedicine utilization. The final number of subjects in the analysis was 18 204 (IDD: n = 6068, non-IDD: n = 12 136 based on 1:2 propensity score matching). The average (SD) age of the subjects was 31 (11.3) years old in 2019. Forty percent of the subjects were female, about 70% of subjects were covered by Medicaid in 2019. Average (SD) number of telemedicine use in 2020 (IDD: 1.96 (5.97), non-IDD: 1.18 (4.90); P < .01) and 2021 (IDD: 2.24 (6.78) vs 1.37 (5.13); P < .01) were higher for the IDD group than the non-IDD group. The regression results showed that the subjects with IDD had 56% more telemedicine encounters than those in the non-IDD group (Incidence Rate Ratio (IRR) = 1.56, P < .01). The growth of telemedicine during the COVID-19 pandemic has the potential to reduce persistent healthcare disparities in individuals with IDD. However, quality of telemedicine should be considered when it is provided to improve health of subjects with IDD.

National outcomes of bridge to multiorgan cardiac transplantation using mechanical circulatory support.

BACKGROUND: Little is known regarding the profile of patients with multiorgan failure listed for simultaneous cardiac transplantation and secondary organ. In addition, few studies have reported how these patients are bridged with mechanical circulatory support (MCS). In this study, we examined national data of patients listed for multiorgan transplantation and their outcomes after bridging with or without MCS. METHODS: United Network for Organ Sharing data were reviewed for adult multiorgan transplantations from 1986 to 2019. Post-transplant patients and total waitlist listings were examined and stratified according to MCS status. Survival was assessed via Cox regression in the post-transplant cohort and Fine-Gray competing risk regression with transplantation as a competing risk in the waitlist cohort. RESULTS: There were 4534 waitlist patients for multiorgan transplant during the study period, of whom 2117 received multiorgan transplants. There was no significant difference in post-transplant survival between the MCS types and those without MCS in the whole cohort and heart-kidney subgroup. Fine-Gray competing risk regression showed that patients bridged with extracorporeal membrane oxygenation had significantly greater waitlist mortality compared with those without MCS when controlling for preoperative characteristics (subdistribution hazard ratio, 2.27; 95% confidence interval, 1.48-3.47; P < .001), whereas those bridged with a ventricular assist device had a decreased incidence of death compared with those without MCS (subdistribution hazard ratio, 0.78; 95% confidence interval, 0.63-0.96; P = .017). CONCLUSIONS: MCS, as currently applied, does not appear to compromise the survival of multiorgan heart transplant patients. Waitlist data show that extracorporeal membrane oxygenation patients have profoundly worse survival irrespective of preoperative factors including organ type listed. Survival on the waitlist for multiorgan transplant has improved across device eras.

Identifying new small proteins through a molecular biology course-based undergraduate research experience laboratory class.

We developed a curriculum for an upper-level molecular biology course-based undergraduate research laboratory class funded by a National Science Foundation CAREER grant that focuses on identifying new small proteins in the bacterium, Escherichia coli. Our CURE class has been continually offered each semester for the last 10 years, with multiple instructors collaboratively developing and implementing their own pedagogical approach while maintaining the same overall scientific goal and experimental strategy. In this paper, we delineate the experimental strategy for our molecular biology CURE laboratory class, describe a range of pedagogical approaches implemented by multiple instructors, and provide recommendations for teaching the class. The purpose of our paper is to share our experiences both in developing and teaching a molecular biology CURE laboratory class based on small protein identification and in creating a curriculum and support system that allows traditional, non-traditional, and under-represented students to participate in authentic research projects.

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast.

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.