Flap Endonuclease Disengages Dna2 Helicase/Nuclease from Okazaki Fragment Flaps

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase {delta}. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing

DECELERATING OPEN CHANNEL FLOW OVER GRAVEL: TURBULENCE STRUCTURE & SENSOR DEVELOPMENT

This dissertation describes investigations of fully turbulent decelerating hydraulically roughbed flow over gravel and the development of technology to measure turbulence and associated sediment transport in streams. Theory is developed for predicting velocity distributions in simple uniform flow using the asymptotic invariance principle and tested using laboratory and field collected data. A mixed scale is developed that accounts for bed derived turbulent structures throughout the flows depth and is used to parameterize the external boundary’s effect on the flow for the logarithmic and outer layers. The asymptotic invariance principle and similarity analysis is conducted for the equations of motion in the outer region of decelerating flow over gravel to define equilibrium conditions for this class of flows with the velocity scale is the freestream velocity. The combination of time series and time averaged statistical analysis of turbulent flow is used to elucidate the structure of flow under decelerating conditions. Time averaged statistical measures of turbulence confirm results of others for higher Froude number approaching transcritical and time series analysis shows the effects of decelerating flow on turbulence to be frequency dependent. Wireless velocity sensors were developed and found capable of measuring time averaged velocity and able to resolve macroturbulence from time series data. A semi-theoretical model of elastic deformation of cantilever beams under hydraulic forcing was coupled with circuit theory to develop a calibration procedure for the VBS that requires only three measurement points, one of which is at zero velocity. Light based sensors are developed to estimate light attenuation in water for ecological research or estimating sediment concentration in water. A semi-theoretical scaling of light attenuation and sediment properties was developed which predicts light attenuation from sediment properties. The combination of new theory on open channel velocity, turbulent structure and field sensors for measuring turbulence and sediment offers the possibility to extend our laboratory knowledge to realistic flow situations

Kinematics of Clustering

The dynamical system for inertial particles in fluid flow has both attracting and repelling regions, the interplay of which can localize particles. In laminar flow experiments we find that particles, initially moving throughout the fluid domain, can undergo an instability and cluster into subdomains of the fluid when the flow Reynolds number exceeds a critical value that depends on particle and fluid inertia. We derive an expression for the instability boundary and for a universal curve that describes the clustering rate for all particles.Comment: 13 pages, 6 figure

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer, which is removed before the fragments are joined. Polymerase d displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Since Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1

Tax research techniques

https://egrove.olemiss.edu/aicpa_guides/1568/thumbnail.jp

Management of Asymptomatic Severe Aortic Stenosis

Patients with symptomatic severe aortic stenosis (AS) benefit from aortic valve replacement surgery, but the management of patients with asymptomatic severe AS is more controversial. While cholesterol and angiotensin have been linked to AS progression, we should await the results of ongoing randomized trials before medical therapy to lower cholesterol or inhibit angiotensin can be recommended to limit disease progression. Clinical factors, echocardiographic parameters, valve morphology, exercise stress testing results, and cardiac biomarkers may be useful in identifying patients who will have early development of symptoms during follow-up and require closer monitoring. The risks associated with aortic valve replacement outweigh the benefits in the majority of patients with asymptomatic severe AS