Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

The DNA replication fork suppresses CMG unloading from chromatin before termination.

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

Structure of the processive human Pol δ holoenzyme.

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 µm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

Initial state of DNA-Dye complex sets the stage for protein induced fluorescence modulation.

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level.

Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1.

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Serum magnesium level and vascular stiffness in children with chronic kidney disease on regular hemodialysis.

Chronic kidney disease (CKD) patients have a high prevalence of vascular calcifications, and cardiovascular disease is the leading cause of death in this population. Magnesium (Mg) depletion may be the missing link between multiple cardiovascular risk factors and the development of atherosclerosis. In this study, we aimed to assess the relationship between serum Mg levels and vascular stiffness in children with CKD on regular hemodialysis (HD). The study included 25 children with CKD on regular HD in our center; the study included also 25 healthy children age-and sex-matched as a control group. Serum Mg levels were measured, and Doppler ultrasound assessment of the intima-media thickness (IMT) and the peak systolic velocities (PSVs) of the main arteries including the (aorta, carotid, and femoral) arteries were recorded in the study patients. There were significantly lower serum Mg levels in children on regular HD than in the controls (1.7 ± 0.43 mg/dL vs. 2.31 ± 0.12 mg/dL, respectively, P = 0.001). There was a significant increase in the aorta and carotid IMT in the study group than in the controls (0.45 ± 0.07 mm vs. 0.40 ± 0.09 mm; 0.98 ± 0.57 mm vs. 0.55 ± 0.1 mm, P = 0.034 and 0.001, respectively), whereas there were no significant differences regarding the PSV of the carotid, aorta, and femoral arteries between the study patients and the controls (P >0.05). A negative correlation was found between serum Mg level with aortic IMT (AIMT) (r = -0.682; P = 0.000). In addition, a significant negative correlation was found between the AIMT with systolic and diastolic blood pressure (r = 0.447, P = 0.025, 0.472, P = 0.017), respectively. We conclude that lower serum Mg levels were associated with vascular calcification in chronic HD children. Confirmation of our results warrants further study.

Case report: hematemesis could be an unusual presentation of cow's milk protein allergy in children in Egypt.

Cow's milk protein allergy (CMPA) is common in infants with variable clinical presentation including varied gastrointestinal manifestation. Cow's milk protein allergy chiefly, involving occurs in children below the age of 3 years, successful therapy depends on completely eliminating cow's milk proteins (CMP) from the child's diet. Ideally, with the replacement of hypo or an allergenic food. Symptoms suggestive of CMPA may be encountered in approximately 5 to 15% of infants emphasizing the importance of controlled elimination/milk challenge procedures. We report on an Egyptian male infant, who developed frequent attacks of hematemesis when begin to eat foods other than breast milk including cow's milk and its dairy products at the age of three months. Possible cow's milk protein allergy was suspected. Further diagnostic work-up was done including: Hb, hematocrit, MCV: iron, ferritin, CRP, occult blood in stools, antibodies to H-pylori and upper GIT endoscopy and biopsy from snip of duodenal mucosa. Measurement of serum cow milk protein specific IgE by radio allegro sorbent test (RAST) technique (immune CAP specific IgE method) and results revealed cow's milk protein allergy. It is concluded that cow's milk protein allergy should be considered in cases of hematemesis presented in early infancy in infants who fed cow's milk early and that hematemesis should be added to the list of clinical presentation of CMPA.

Complete genome sequence of Mycobacterium fortuitum subsp. fortuitum type strain DSM46621.

Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.

Relation between anemia and blood levels of lead, copper, zinc and iron among children.

BACKGROUND: Anemia is a health problem among infants and children. It is often associated with a decrease in some trace elements (iron, zinc, copper) and an increase in heavy metals as lead. This study was done to determine the association of blood lead level > 10 mug/dl, with the increased risk to anemia, also, to investigate the relationship between anemia and changes in blood iron, zinc and copper levels, and measure lead level in drinking water.The study is a cross-sectional performed on 60 children. Venous blood samples were taken from the studied population for estimating hematological parameters as well as iron and ferritin levels. The concentrations of zinc, copper, and lead were measured. The studied population was divided into anemic and non-anemic (control) groups. The anemic group was further classified into mild, moderate and severe anemia. The study subjects were also categorized into low and high blood lead level groups. FINDINGS: Approximately 63.33% of children had blood lead levels >/= 10 mug/dl. At the blood lead level range of 10-20 mug/dl, a significant association was found for mild and severe anemia. The blood level of iron and ferritin was found to be significantly lower in high blood lead level and anemic groups than those of the low blood lead level and control groups. Lead level in drinking water was higher than the permissible limit. CONCLUSION: Lead level >/= 10 mug/dl was significantly associated with anemia, decreased iron absorption and hematological parameters affection. High blood lead levels were associated with low serum iron and ferritin. Lead level in drinking water was found to be higher than the permissible limits.

CD64 cell surface expression on neutrophils for diagnosis of neonatal sepsis.

Neonatal sepsis (NS) continues to be one of the most significant causes of neonatal morbidity and mortality. Early identification of Neonatal sepsis is a major diagnostic problem because of the nonspecific clinical signs and limitations of the current diagnostic procedures. Neutrophil CD64 expression has been proposed as a diagnostic test for evaluation of infection and sepsis. We compared the diagnostic utility of neutrophil CD64 expression with IL-6, IL-8, TNFalpha and CRP assays. Peripheral blood samples were taken from 25 neonates classified into two groups; proven NS (n = 15), clinical NS (n = 10) and healthy newborns (n = 10). CD64 expression was analysed by flowcytometry, while serum level of interleukins (IL-6, IL-8), and TNFalpha was determined by ELISA. Expression of CD64 was significantly enhanced in the groups with proven sepsis and clinical NS as compared to the controls (P < 0.05). Similary, TNFalpha, IL-6, IL-8 and CRP levels were significantly elevated in the groups with sepsis and clinical NS as compared to the controls (P < 0.05). Our data indicate that, in addition to serum levels of interleukins (IL-6, IL-8), and TNFalpha, expression of CD64 on neutrophils by flowcytometry could be useful as an indicator of NS due to its early appearance, sensitivity and specificity (96%). In conclusion, neutrophil expression of CD64 is a useful diagnostic tool for early detection of neonatal sepsis. The assay is rapid, easy and reliable.