DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

Estimating Photosynthesis and Concurrent Export Rates in C3 and C4 Species at Ambient and Elevated CO21 2

The ability of 21 C3 and C4 monocot and dicot species to rapidly export newly fixed C in the light at both ambient and enriched CO2 levels was compared. Photosynthesis and concurrent export rates were estimated during isotopic equilibrium of the transport sugars using a steady-state 14CO2-labeling procedure. At ambient CO2 photosynthesis and export rates for C3 species were 5 to 15 and 1 to 10 μmol C m−2 s−1, respectively, and 20 to 30 and 15 to 22 μmol C m−2 s−1, respectively, for C4 species. A linear regression plot of export on photosynthesis rate of all species had a correlation coefficient of 0.87. When concurrent export was expressed as a percentage of photosynthesis, several C3 dicots that produced transport sugars other than Suc had high efflux rates relative to photosynthesis, comparable to those of C4 species. At high CO2 photosynthetic and export rates were only slightly altered in C4 species, and photosynthesis increased but export rates did not in all C3 species. The C3 species that had high efflux rates relative to photosynthesis at ambient CO2 exported at rates comparable to those of C4 species on both an absolute basis and as a percentage of photosynthesis. At ambient CO2 there were strong linear relationships between photosynthesis, sugar synthesis, and concurrent export. However, at high CO2 the relationships between photosynthesis and export rate and between sugar synthesis and export rate were not as strong because sugars and starch were accumulated.

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism.

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and -pulsed folding and annealing cage.

Free GroEL binds denatured proteins very tightly: it retards the folding of barnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight-binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES.nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES.nucleotide complex. Fast-folding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL.denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL.GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination of folding cage and an annealing machine.

Estimación del movimiento coherente: computación evolutiva como alternativa al annealing determinista

Comunicación presentada en SCETA, Seminario Sobre Computación Evolutiva, celebrado en la VII Conferencia de la Asociación Española para la Inteligencia Artificial, CAEPIA, Málaga, 12-14 noviembre 1997.

Mechanical annealing of metallic electrodes at the atomic scale

The process of creating an atomically defined and robust metallic tip is described and quantified using measurements of contact conductance between gold electrodes and numerical simulations. Our experiments show how the same conductance behavior can be obtained for hundreds of cycles of formation and rupture of the nanocontact by limiting the indentation depth between the two electrodes up to a conductance value of approximately 5G0 in the case of gold. This phenomenon is rationalized using molecular dynamics simulations together with density functional theory transport calculations which show how, after repeated indentations (mechanical annealing), the two metallic electrodes are shaped into tips of reproducible structure. These results provide a crucial insight into fundamental aspects relevant to nanotribology or scanning probe microscopies.

Retrofit of heat exchanger networks with pressure recovery of process streams at sub-ambient conditions

This paper presents a new mathematical programming model for the retrofit of heat exchanger networks (HENs), wherein the pressure recovery of process streams is conducted to enhance heat integration. Particularly applied to cryogenic processes, HENs retrofit with combined heat and work integration is mainly aimed at reducing the use of expensive cold services. The proposed multi-stage superstructure allows the increment of the existing heat transfer area, as well as the use of new equipment for both heat exchange and pressure manipulation. The pressure recovery of streams is carried out simultaneously with the HEN design, such that the process conditions (streams pressure and temperature) are variables of optimization. The mathematical model is formulated using generalized disjunctive programming (GDP) and is optimized via mixed-integer nonlinear programming (MINLP), through the minimization of the retrofit total annualized cost, considering the turbine and compressor coupling with a helper motor. Three case studies are performed to assess the accuracy of the developed approach, including a real industrial example related to liquefied natural gas (LNG) production. The results show that the pressure recovery of streams is efficient for energy savings and, consequently, for decreasing the HEN retrofit total cost especially in sub-ambient processes.

Organic distributed feedback laser to monitor solvent extraction upon thermal annealing in solution-processed polymer films

Solution-processed polymer films are used in multiple technological applications. The presence of residual solvent in the film, as a consequence of the preparation method, affects the material properties, so films are typically subjected to post-deposition thermal annealing treatments aiming at its elimination. Monitoring the amount of solvent eliminated as a function of the annealing parameters is important to design a proper treatment to ensure complete solvent elimination, crucial to obtain reproducible and stable material properties and therefore, device performance. Here we demonstrate, for the first time to our knowledge, the use of an organic distributed feedback (DFB) laser to monitor with high precision the amount of solvent extracted from a spin-coated polymer film as a function of the thermal annealing time. The polymer film of interest, polystyrene in the present work, is doped with a small amount of a laser dye as to constitute the active layer of the laser device and deposited over a reusable DFB resonator. It is shown that solvent elimination translates into shifts in the DFB laser wavelength, as a consequence of changes in film thickness and refractive index. The proposed method is expected to be applicable to other types of annealing treatments, polymer-solvent combinations or film deposition methods, thus constituting a valuable tool to accurately control the quality and reproducibility of solution-processed polymer thin films.