We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.
The excimer-forming fluorophore dipyrenylpropane has been used to measure the relative fluidity of total membranes isolated from Escherichia coli grown at 30 or 45 degrees C, or exposed to a heat-shock from 30 to 45 degrees C for various periods of time. Parallel experiments were performed using [35S]methionine pulse-labeling of cells, to study the induction of heat-shock proteins (HSPs) at different times after the sudden change in E. coli growth-temperature from 30 to 45 degrees C. Results suggest that upon an abrupt temperature upshift from 30 to 45 degrees C, membrane fluidity adjustment to the steady-state level at the high temperature, takes place during the E. coli heat-shock response.
The possible changes in the fatty acid profile of Escherichia coli during heat‐shock have been investigated. Bacteria growing in steady‐state at 30°C were subjected to an abrupt temperature upshift to 45°C and held at the high temperature for various periods of time in order to elicit the heat‐shock response. Fatty acid compositions of lipids extracted from samples taken at different times after the temperature upshift, as well as from cultures in steady‐state at 30 and 45°C, were determined by gas‐chromatography. It has been found that the total unsaturates to total saturates ratio decreases gradually during heat‐shock and that 30 min after the temperature jump, the reduction is equivalent to 57% of the difference between ratios corresponding to steady‐state cultures at 30 and 45°C. Consistent with this remodeling of lipid acyl chains, there is a decrease in the excimerization rate of the fluidity probe dipyrenylpropane incorporated into sonicated E. coli lipid extracts. Such modifications occur within the time‐span of the heat‐shock response, as judged from our previous measurements of the kinetics of change in heat‐shock proteins induction ratio. Together, these results indicate that the control of membrane fluidity during the heat‐shock response can be accounted for, at least in part, by an important change in the fatty acid composition of Escherichia coli lipids.
Analysis of the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes catalyzed by porcine pancreatic phospholipase A2 at 18 degrees C shows that, in the presence of 10 mM NaCl, the length of the latency period in the presteady-state phase increases from 3 to 10.5 min when the CaCl2 concentration is reduced from 15 to 1 mM. This inverse dependence of the lag period on calcium ion concentration is seen more readily at 1 M NaCl, where the induction time changes from 13.5 to 42 min by decreasing the concentration of CaCl2 from 15 to 1 mM. To interpret these results, we took into account the small amount of fatty acid that is produced during the latency phases. The fatty acid generates a negative surface electrostatic potential and makes the interfacial concentration of calcium ions different from the concentration in the bulk solvent. Variations in the analytical concentrations of NaCl and CaCl2 affect both the interfacial calcium ion concentration and electrostatic potential, as estimated theoretically from Grahame and Boltzmann equations. According to these estimates, the length of the latency period diminishes with the increase of the interfacial calcium concentration, but does not show any logical dependence on the change in surface electrostatic potential.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.