The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition.

The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.     

An overview of homologous pairing and DNA strand exchange proteins.

Processes fundamental to all models of genetic recombination include the homologous pairing and subsequent exchange of DNA strands. Biochemical analysis of these events has been conducted primarily on the recA protein of Escherichia coli, although proteins which can promote such reactions have been purified from many sources, both prokaryotic and eukaryotic. The activities of these homologous pairing and DNA strand exchange proteins are either ATP-dependent, as predicted based on the recA protein paradigm, or, more unexpectedly, ATP-independent. This review examines the reactions promoted by both classes of proteins and highlights their similarities and differences. The mechanistic implications of the apparent existence of 2 classes of strand exchange protein are discussed.     

Enhancement of Escherichia coli RecA protein enzymatic function by dATP

The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA. The substitution of dATP for rATP has significant effects on various recA protein biochemical properties. In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rATP. The dATP-recA protein complex can compete more effectively with the E. coli ssDNA binding protein (SSB) for ssDNA binding sites compared with the rATP-recA protein complex. Finally, the rate of dATP hydrolysis stimulated by dsDNA is greater than the rate of rATP hydrolysis. These effects, in turn, are observed as alterations in the recA protein catalyzed DNA strand exchange reaction. In the absence of SSB protein, the rate of joint molecule and product formation in the DNA strand exchange reaction is greater in the presence of dATP than in the presence of rATP. The rate of product formation in the dATP-dependent reaction is also faster than the rATP-dependent reaction when SSB protein is added to the ssDNA before recA protein; the rate of rATP-dependent product formation is inhibited 10-fold under these conditions. This nucleotide, dATP, was previously shown to induce an apparent affinity of recA protein for ssDNA which is higher than any other NTP. These results suggest that the observed enhancement of enzymatic activity may be related to the steady-state properties of the high-affinity ssDNA binding state of recA protein. In addition, the data suggest that recA protein functions in NTP hydrolysis as a dimer of protein filaments and that the binding of ssDNA to only one of the recA filaments is sufficient to activate all recA protein molecules in the dimeric filament. The implications of this finding to the enzymatic function of recA protein are discussed.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.     

An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

Rad54 protein is a key member of the RAD52 epistasis group required for homologous recombination in eukaryotes. Rad54 is a duplex DNA translocase that remodels both DNA and protein–DNA complexes, and functions at multiple steps in the recombination process. Here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. The Sulfolobus solfataricus Rad54 (SsoRad54) protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the S. solfataricus Rad51 homologue, SsoRadA, to stimulate DNA strand exchange. Confirmation of this protein as an authentic Rad54 homolog establishes an essential phylogenetic bridge for identifying Rad54 homologs in the archaeal and bacterial domains.