An in vitro DNA phosphorothioate modification reaction.

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

Phosphorothioated DNA Is Shielded from Oxidative Damage.

DNA is the carrier of genetic information. DNA modifications play a central role in essential physiological processes. Phosphorothioation (PT) modification involves the replacement of an oxygen atom on the DNA backbone with a sulfur atom. PT modification can cause genomic instability in Salmonella enterica under hypochlorous acid stress. This modification restores hydrogen peroxide (H2O2) resistance in the catalase-deficient Escherichia coli Hpx- strain. Here, we report biochemical characterization results for a purified PT modification protein complex (DndCDE) from S. enterica We observed multiplex oligomeric states of DndCDE by using native PAGE. This protein complex bound avidly to PT-modified DNA. DndCDE with an intact iron-sulfur cluster (DndCDE-FeS) possessed H2O2 decomposition activity, with a Vmax of 10.58 ± 0.90 mM min-1 and a half-saturation constant, K0.5S, of 31.03 mM. The Hill coefficient was 2.419 ± 0.59 for this activity. The protein's activity toward H2O2 was observed to be dependent on the intact DndCDE and on the formation of an iron-sulfur (Fe-S) cluster on the DndC subunit. In addition to cysteine residues that mediate the formation of this Fe-S cluster, other cysteine residues play a catalytic role. Finally, catalase activity was also detected in DndCDE from Pseudomonas fluorescens Pf0-1. The data and conclusions presented suggest that DndCDE-FeS is a short-lived catalase. Our experiments also indicate that the complex binds to PT sites, shielding PT DNA from H2O2 damage. This catalase shield might be able to extend from PT sites to the entire bacterial genome.IMPORTANCE DNA phosphorothioation has been reported in many bacteria. These PT-hosting bacteria live in very different environments, such as the human body, soil, or hot springs. The physiological function of DNA PT modification is still elusive. A remarkable property of PT modification is that purified genomic PT DNA is susceptible to oxidative cleavage. Among the oxidants, hypochlorous acid and H2O2 are of physiological relevance for human pathogens since they are generated during the human inflammation response to bacterial infection. However, expression of PT genes in the catalase-deficient E. coli Hpx- strain restores H2O2 resistance. Here, we seek to solve this obvious paradox. We demonstrate that DndCDE-FeS is a short-lived catalase that binds tightly to PT DNA. It is thus possible that by docking to PT sites the catalase activity protects the bacterial genome against H2O2 damage.

Grayscale ultrasonic and shear wave elastographic characteristics of the Achilles' tendon in patients with familial hypercholesterolemia: A pilot study.

OBJECTIVE: To investigate the feasibility of grayscale ultrasound and quantitative shear wave elastography (SWE) for assessing the image features and stiffness of the Achilles tendon in patients with familial hypercholesterolemia (FH) compared with healthy controls. METHODS: A total of 38 Achilles tendons from healthy control participants and 94 from patients with FH were examined with grayscale ultrasound and SWE. Each Achilles tendon examination was performed on 3 different segments (proximal, middle, and distal). The thickness, grayscale image features and elasticity modulus of each segment was measured on longitudinal views. RESULTS: In Achilles tendons of healthy control participants, the thicknesses of the proximal, middle, and distal segments were (4.46 ± 0.68) mm, (5.41 ± 1.01) mm, and (4.41 ± 0.57) mm, respectively. The grayscale features were diffusely hypoechoic with parallel linear hyperechoic striations. The mean elasticity modulus of the proximal, middle, and distal sites was (418.08 ± 43.13) kPa, (425.78 ± 47.14) kPa, and (407.79 ± 38.74) kPa, respectively. In the FH group, the thicknesses of the proximal, middle, and distal segments were (7.65 ± 3.09) mm, (11.46 ± 4.84) mm, and (8.14 ± 2.90) mm, respectively. The grayscale features were hypoechoic with linear hyperechoic disordered fibre distribution (92/94). Two of 94 Achilles tendons had considerable focal hypoechogenicity. Seventeen of 94 Achilles tendons had calcifications. In the FH group, the mean elasticity modulus of the proximal, middle, and distal segments were (294.86 ± 58.13) kPa, (280.93 ± 63.58) kPa, and (282.41 ± 56.47) kPa, respectively. Statistically significant differences were found between the Achilles tendons of healthy control participants and FH patients in the thicknesses and mean elasticity modulus at the proximal, middle and distal segments of the Achilles tendons (all P < 0.001). CONCLUSIONS: Our results suggest that the grayscale features and SWE characteristics of the Achilles tendon provided complementary biomechanical information for quantitative assessment the Achilles tendon in patients with FH.