DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP

The Rad50, Mre11 and Nbs1 complex is involved in many essential chromosomal organization processes dealing with DNA ends, including two major pathways of DNA double-strand break repair, homologous recombination and non-homologous end joining. Previous data on the structure of the human Rad50 and Mre11 (R/M) complex suggest that a common role for the protein complex in these processes is to provide a physical link between DNA ends such that they can be processed in an organized and coordinated manner. Here we describe the DNA binding properties of the R/M complex. The complex bound to both single-stranded and double-stranded DNA. Scanning force microscopy analysis of DNA binding by R/M showed the requirement for an end to form oligomeric R/M complexes, which could then migrate or transfer away from the end. The R/M complex had a lower preference for DNA substrates with 3′-overhangs compared with blunt ends or 5′-overhangs. Interestingly, ATP binding, but not hydrolysis, increased the preference of R/M binding to DNA substrates with 3′-overhangs relative to substrates with blunt ends and 5′-overhangs.     

A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release

Lysophosphatidylcholines(lyso-PCs), generated during blood storage, are etiologic in atwo-insult, sepsis-based model of transfusion-related acute lung injury(TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do notactivate neutrophils (PMNs). We hypothesized that priming of PMNsalters their reactivity such that a second priming agent causes PMNactivation and endothelial cell damage. PMNs were primed or not withLPS and then treated with lyso-PCs, and oxidase activation and elastaserelease were measured. For coculture experiments, activation of humanpulmonary microvascular endothelial cells (HMVECs) was assessed byICAM-1 expression and chemokine release. HMVECs were stimulated ornot with LPS, PMNs were added, cells were incubated with lyso-PCs, andthe number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 andrelease of ENA-78, GRO, and IL-8. PMN-mediated HMVEC damage wasdependent on LPS activation of HMVECs, chemokine release, PMNadhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates themicrobicidal arsenal, and PMN-mediated HMVEC damage was the resultof two insults: HMVEC activation and PMN oxidase assembly.

     

Experimental assessment of salamanders as predators of detrital food webs: effects on invertebrates, decomposition and the carbon cycle

Declines in amphibian populations and the loss of some species has spurred research into the causes of declines. Little research has been done on what the loss of amphibian species may mean to ecosystem function. I describe a field experiment where the number of Plethodon cinereus (the red-backed salamander) was manipulated in enclosures buried in the forest floor. I determined the composition of the forest floor invertebrate community and the rate of decomposition of leaf litter in enclosures with and without salamanders over 4.5 months. Salamander predation in enclosures significantly reduced invertebrate numbers compared to control enclosures. Salamander predation also indirectly reduced rate of decomposition by between 11 and 17%. This was probably due to salamanders consuming a significant number of leaf fragmenters (e.g. Coleoptera and Diptera larvae). Predation on leaf litter fragmenters may alter carbon dynamics in forested ecosystems.     

Modification of the Fe Protein of Nitrogenase in Natural Populations of Trichodesmium thiebautii

The Fe protein of nitrogenase in the marine nonheterocystous cyanobacterium Trichodesmium thiebautii is interconverted between two forms, which is reminiscent of the ADP-ribosylation described in the purple bacterium Rhodospirillum rubrum. In natural populations of T. thiebautii during the day, when nitrogenase activity (NA) is present and while photosynthetic rates are high, a low-molecular-mass form of the Fe protein is present. In the late afternoon, the low-molecular-mass form is partially converted to a higher-molecular-mass form (approximately equal distribution of high- and low-molecular-mass forms of the Fe protein subunits), concurrent with cessation of NA. Some of the higher-molecular-mass form persists through the night until the very early morning, when the lower-molecular-mass form appears. New synthesis of both the Fe and MoFe proteins of nitrogenase appears to occur at this time. The higher-molecular-mass form of the Fe protein is also produced rapidly in response to artificially elevated external O₂ levels (40%) during the day. T. thiebautii is capable of recovery of NA in less than 1 h following exposure to 40% O₂, which is correlated with the return of the Fe protein to the lower-molecular-mass form. Recovery from exposure to O₂ is not dependent upon protein synthesis. The modification of the Fe protein is clearly involved in regulation of NA during the diel cycle of NA in T. thiebautii but may also be involved in protecting the Fe protein during transient O₂ concentration increases.     

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophylla/c-containing protein from the chrysophyteIsochrysis galbana: implications for evolution of thecab gene family

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyteIsochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophylla/c-binding light-harvesting protein (FCP) ofI. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chla/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity betweenI. galbana FCP and othercab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related tocab genes encoding LHC I, CP 29, and CP 24 of higher plants than tocab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chla/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.     

Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.     

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.     

Torque-limited RecA polymerization on dsDNA

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA–DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 ± 0.7 k_bT. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.     

Assembly and function of the Photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404–414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S–S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta 1274: 135–142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the pI of the MSPs and BSA enhanced their aggregation resistance, a behavior which can be predicted from their charge (MSP) or a combination of charge and stabilization by -S–S- bonds (BSA). In the case of aggregation resistance by MSP, this is likely to be an important factor in its ability to avoid unproductive self-association reactions in favor of formation of the protein–protein interactions that lead to formation of the functional oxygen evolving complex.     

Structure and Activity of the Photosystem II Manganese-Stabilizing Protein: Role of the Conserved Disulfide Bond

The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme’s active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins [Lydakis-Simantiris et al. (1999b) Biochemistry 38: 404–414]. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution [Shutova et al. (2000) FEBS Lett. 467: 137–140]. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O₂ evolution activity and binds to MSP-free PS II preparations at wild-type levels [Betts et al. (1996) Biochim. Biophys. Acta 1274: 135–142]. This mutant was further characterized by incubation at 90 °C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90 °C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O₂ evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O₂ evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803 [Burnap et al. (1994) Biochemistry 33: 13712–13718].