The rotation-coupled sliding of EcoRV

It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE–DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D1), revealing a significant size dependency. To account for the dependence of D1 on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts

The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5'-CC/SGG-3' (where S = C or G, ‘/’ designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level.We find, using FRET, that BcnI adopts either ‘open’ or ‘closed’ conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI–DNA interactions

Effect of astaxanthin supplementation on muscle damage and oxidative stress markers in elite young soccer players

Aim. The purpose of the current study was to examine the effect of Astaxanthin (Asx) supplementation on muscle enzymes as indirect markers of muscle damage, oxidative stress markers and antioxidant response in elite young soccer players. Methods. Thirty-two male elite soccer players were randomly assigned in a double-blind fashion to Asx and placebo (P) group. After the 90 days of supplementation, the athletes performed a 2 hour acute exercise bout. Blood samples were obtained before and after 90 days of supplementation and after the exercise at the end of observational period for analysis of thiobarbituric acid-reacting substances (TBARS), advanced oxidation protein products (AOPP), superoxide anion (O-2(center dot-)), total antioxidative status (TAS), sulphydril groups (SH), superoxide-dismutase (SOD), serum creatine kinase (CK) and aspartate aminotransferase (AST). Results. TBARS and AOPP levels did not change throughout the study. Regular training significantly increased O-2(center dot-) levels (main training effect, P lt 0.01). O-2(center dot-) increased after the soccer exercise (main exercise effect, P lt 0.01), but these changes reached statistical significance only in the P group (exercise x supplementation effect, P lt 0.05). TAS levels decreased significantly post- exercise only in P group (P lt 0.01). Both Asx and P groups experienced increase in total SH groups content (by 21% and 9%, respectively) and supplementation effect was marginally significant (P=0.08). Basal SOD activity significantly decreased both in P and in Asx group by the end of the study (main training effect, P lt 0.01). All participants showed a significant decrease in basal CK and AST activities after 90 days (main training effect, P lt 0.01 and P lt 0.001, respectively). CK and AST activities in serum significantly increased as result of soccer exercise (main exercise effect, P lt 0.001 and P lt 0.01, respectively). Postexercise CK and AST levels were significantly lower in Asx group compared to P group (P lt 0.05) Conclusion. The results of the present study suggest that soccer training and soccer exercise are associated with excessive production of free radicals and oxidative stress, which might diminish antioxidant system efficiency. Supplementation with Asx could prevent exercise induced free radical production and depletion of non-enzymatic antioxidant defense in young soccer player