Human Rad52 binding renders ssDNA unfolded: image and contour length analyses by Atomic Force Microscopy

Atomic force microscopy imaging has been used to study the changes associated with human Rad52 (HsRad52) protein in solution, in dried state as well as following ssDNA (linear and circular) binding. In the dried state, the free protein exists predominantly as a characteristic panoply of novel trifoliate forms. However, in solution, the level of trifoliates diminishes significantly. Height analyses of either form reveal two categories: smaller (~ 3-5 nm) and larger ((~ 10-12 nm) particles, perhaps related to sub-heptameric and heptameric forms respectively. Interestingly, binding of the protein to linear ssDNA smoothly extends and unfolds the naked DNA. Contour length measurements performed on several individual circular ssDNA/nucleoprotein complexes reveal marked (about threefold) extension of naked ssDNA, following HsRad52 binding. We speculate that the alignment of HsRad52 on ssDNA into a smoothly extended and unfolded strand from that of highly compact morphology of naked ssDNA, may have bearing on the recombination function of HsRad52 protein

Hormonal regulation of moss protonema development and the possible origin of plant hormonal responses in bryophytes

The protonema of mosses is a far simpler paradigm to understand the mechanism of hormonal action and tolerance to abiotic stresses in plants. Its developmental biology, responses to hormones and the similarity of signaling mechanisms with higher plants are reviewed. There is strong evidence for second messenger role of calcium ions in the action of cytokinin. Multiple calcium-dependent protein kinases (CDPKs) are present in the protonema. The Funaria hygrometrica CDPK gene (FhCDPK) shows the characteristic catalytic and autoinhibitory domains, the four EF hands and the highest homology to CDPKs from higher plants but far lower to liverwort or other moss CDPK genes. A 38 kDa myelin basic protein kinase (MBP kinase) is activated within minutes by abscisic acid (ABA) and salinity. As ABA also confers tolerance against desiccation and freezing and the wheat ABA-inducible promoter is fully functional in mosses, the ABA signaling mechanism seems to be highly conserved. In plants, the CDPKs are involved not only in hormonal signaling but also in the acclimation response against abiotic stresses. The manipulation of signal transduction components such as transcription factors, CDPKs and calcineurin have emerged as viable strategies to genetically engineer the stress tolerant plants. There is increasing evidence to support the origin of plant hormonal responses at the level of bryophytes

Hormonal Regulation of Moss Protonema Development and the Possible Origin of Plant Hormonal Responses in Bryophytes

9-16The protonema of mosses is a far simpler paradigm to understand the mechanism of hormonal action and tolerance to abiotic stresses in plants. Its developmental biology, responses to hormones and the similarity of signaling mechanisms with higher plants are reviewed. There is strong evidence for second messenger role of calcium ions in the action of cytokinin. Multiple calcium-dependent protein kinases (CDPKs) are present in the protonema. The Funaria hygrometrica CDPK gene (FhCDPK) shows the characteristic catalytic and autoinhibitory domains, the four EF hands and the highest homology to CDPKs from higher plants but far lower to liverwort or other moss CDPK genes. A 38 kDa myelin basic protein kinase (MBP kinase) is activated within minutes by abscisic acid (ABA) and salinity. As ABA also confers tolerance against desiccation and freezing and the wheat ABA-inducible promoter is fully functional in mosses, the ABA signaling mechanism seems to be highly conserved. In plants, the CDPKs are involved not only in hormonal signaling but also in the acclimation response against abiotic stresses. The manipulation of signal transduction components such as transcription factors, CDPKs and calcineurin have emerged as viable strategies to genetically engineer the stress tolerant plants. There is increasing evidence to support the origin of plant hormonal responses at the level of bryophytes

Ca<SUP>2+</SUP> dPKs from the protonema of the moss Funaria hygrometrica: effect of indole-acetic acid and cultural parameters on the activity of a 44 kDa Ca<SUP>2+</SUP> dPK

Using the in-gel kinase assay, four polypeptides of Mr 44, 48, 63, and 70 kDa showed calcium-dependent phosphorylation in the crude extract of the moss Funaria hygrometrica Hedw. Analysis by SDS-PAGE suggests that these phosphopolypeptides represent the calcium-dependent protein kinases (PK), which could undergo autophosphorylation and phosphorylate casein. Temporal changes in the activity of PK in cells grown under different conditions were detectable using the in-gel kinase assay. The 44 kDa Ca2+ dPK showed an increase in activity in cells grown either in the presence of auxin, at pH 5, or when starved of nitrate. This PK is shown to share epitopes with calmodulin suggesting that it could be a PK with calmodulin-like domain. The in-gel kinase assay, its possible applications and the existence of multiple Ca2+ dPKs in moss are discussed

Purification and characterization of a Ca²⁺-dependent/calmodulin-stimulated protein kinase from moss chloronema cells

We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the mossFunaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 (AM free Ca2+, and was further enhanced 3-5-fold with exogenously added 3-1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Underin vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK

ABA and NaCl activate myelin basic protein kinase in the chloronema cells of the moss Funaria hygrometrica

Mitogen-activated protein kinases (MAPKs) are components of highly conserved signal transduction pathways. We show that in pH 5-grown chloronema cells of the moss Funaria hygrometrica, abscisic acid (ABA) and NaCl caused a rapid increase in the activity of a 38 kDa protein kinase (PK38) that uses myelin-basic protein (MBP) as a substrate. The activity was dose-dependent, and seemed to saturate with 20 μ M ABA or 200 mM NaCl. It was also strongly stimulant-dependent, as the withdrawal of ABA or NaCl reduced the activity drastically. PK38 also utilized a synthetic peptide specific for extracellular-regulated kinases (ERKs: EC 2.7.1.37). The responses to NaCl and ABA were additive, suggesting that the two stimuli act via independent, parallel pathways. The cation requirement and ERK substrate specificity suggest that PK38 could be a stress-activated PK

GTP-induced conformational changes in translin: a comparison between human and Drosophila proteins

Human translin is a conserved protein, unique in its ability to bind both RNA and DNA. Interestingly, GTP binding has been implicated as a regulator of RNA/DNA binding function of mouse translin (TB-RBP). We cloned and overexpressed the translin orthologue from Drosophila melanogaster and compared its DNA/RNA binding properties in relation to GTP effects with that of human protein. Human translin exhibits a stable octameric state and binds ssDNA/RNA/dsDNA targets, all of which get attenuated when GTP is added. Conversely, Drosophila translin exhibits a stable dimeric state that assembles into a suboctameric (tetramer/hexamer) form and fails to bind ssDNA and RNA targets. Interestingly enough, CD spectral analyses, partial protease digestion profile revealed GTP-specific conformational changes in human translin, whereas the same were largely missing in Drosophila protein. Isothermal calorimetry delineated specific heat changes associated with GTP binding in human translin, which invoked subunit “loosening” in its octamers; the same effect was absent in Drosophila protein. We propose that GTP acts as a specific molecular “switch” that modulates the nucleic acid binding function selectively in human translin, perhaps by affecting its octameric configuration

Sensitive, real-time monitoring of UV-induced stress in a single, live plant cell using an optical trap

An optical trap is used to monitor bio-deleterious effects induced by ultra-violet (UV) radiation in a single, live cell. When placed in a laser optical trap, Chlamydomonas reinhardtii cells undergo rotation because of the interplay of flagellar-based forces and optical forces generated by the laser light. Such rotation is sustained by the trapped cell's flagellar action, thereby unveiling a robust and quantitative cellular assay for flagellar function. UV induces flagellar damage, leading to dose-dependent attenuation of cellular rotation. At UV doses larger than ∃ 9 J m<SUP>-2</SUP> rotational motion ceases. The ability of the cell to rotate in an optical trap is, therefore, a measure of the damage response of the cell. By monitoring cell rotation, we can quantify UV damage with high sensitivity, at threshold doses as low as 2 J m<SUP>-2</SUP>. Apart from the capability of UV-damage detection, our rotation assay is also sensitive to the cellular protective responses against such damage. To illustrate this additional facet, we quantify the efficacy of ascorbic acid in combating the UV-damage response of individual cells. Upon addition of this antioxidant, there is no cessation of rotations for doses as high as 25 J m<SUP>-2</SUP>. This represents the first single-cell sensor that robustly quantifies a complex pleiotropic cellular response