Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2-3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 > Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT](10) and [TC](11) microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo

In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.     

h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.     

Effect of DNA polymerase beta loop variants on discrimination of O6-methyldeoxyguanosine modification present in the nucleotide versus template substrate

We have examined the mechanism of DNA polymerase beta (pol beta) lesion discrimination using alkylated dNTP versus alkylated DNA template substrates and the pol beta variants R253M and E249K. Both of these amino acid variants are located in the loop region of the palm domain and are known to play a role in pol beta fidelity and discrimination of 3'-azido-3'-deoxythymidine triphosphate substrates. We observed that these variants affect O(6)-methyldeoxyguanosine- (m6G-) modified dNTP discrimination without affecting m6G template translesion synthesis. Under steady-state conditions, the ratio of inherent reactivity values for the m6dGTP substrate relative to the dGTP substrate was greater for both variant polymerases than for wild-type (WT) pol beta. Biochemical assays of translesion synthesis using m6G lesion-containing templates demonstrated no significant differences between the variants and WT. Using N-methyl-N-nitrosourea- (MNU-) modified DNA templates in the HSV-tk in vitro assay, no difference among the enzymes in the frequency of alkylation-induced G to A transition mutations was observed. However, differences among the polymerases in the frequency of alkylation-induced C to A transversions were observed, consistent with a mutator tendency for E249K and an antimutator tendency for R253M. We conclude that a specific interaction at the loop of the palm domain is involved in pol beta discrimination of the m6G lesion when present on the incoming dNTP substrate but not when present in the DNA template. Our data support a role for the flexible loop in pol beta error discrimination.     

Evolution of self-fertilization at geographical range margins? A comparison of demographic, floral, and mating system variables in central vs. peripheral populations of Aquilegia canadensis (Ranunculaceae)

Biogeographic models predict that geographically peripheral populations should be smaller, more sparsely distributed, and have a lower per-capita reproductive rate than populations near the center of a species' range. Plants in peripheral populations may, therefore, receive less pollinator visitation and outcross pollination, which may select for self-fertilization to provide reproductive assurance. We tested these predictions by comparing population size, plant density, seed production, floral traits, and mating system parameters between 10 populations of Aquilegia canadensis near the northern margin of the range with 10 near the range center. Contrary to predictions, peripheral populations were not smaller, less dense, nor less productive than central populations. Nevertheless, we detected substantial regional differences in key floral traits. Plants in central populations produced larger flowers with 68% greater herkogamy and had 30% more flowers open simultaneously than plants in northern populations. However, there was no regional difference in the mating system. In northern populations, 73% (range = 60-88%) of seeds were self-fertilized compared to 76% (51-100%) in central populations. In both regions, adult inbreeding coefficients were near zero, indicating very strong inbreeding depression despite high selfing. Marked geographic variation in key floral traits does not reflect evolutionary differentiation in the mating system.     

Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant-negative effect on the wild-type protein in HeLa cells

To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.     

Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis

Type I transglutaminase (TG1) is an enzyme that is responsible for assembly of the keratinocyte cornified envelope. Although TG1 mutation is an underlying cause of autosomal recessive congenital ichthyosis, a debilitating skin disease, the pathogenic mechanism is not completely understood. In the present study we show that TG1 is an endoplasmic reticulum (ER) membrane-associated protein that is trafficked through the ER for ultimate delivery to the plasma membrane. Mutation severely attenuates this processing and a catalytically inactive point mutant, TG1-FLAG(C377A), accumulates in the endoplasmic reticulum and in aggresome-like structures where it is ubiquitinylated. This accumulation results from protein misfolding, as treatment with a chemical chaperone permits it to exit the endoplasmic reticulum and travel to the plasma membrane. ER accumulation is also observed for ichthyosis-associated TG1 mutants. Our findings suggest that misfolding of TG1 mutants leads to ubiquitinylation and accumulation in the ER and aggresomes, and that abnormal intracellular processing of TG1 mutants may be an underlying cause of ichthyosis.     

Alterations in delayed and direct phytoplankton fluorescence in response to the diurnal light cycle

Delayed fluorescence (DF) excitation spectrometry was examined as a proxy for phytoplankton activity in comparison to pulse-amplitude-modulated (PAM) fluorometry and dissolved oxygen (DO) evolution. During several day–night cycles, the three target variables were monitored simultaneously, together with pH, temperature and photosynthetically active irradiance in an exponentially growing Chlorella population exposed to natural light conditions. It was found that during a diel cycle prompt and DF signals corresponded to each other and were negatively correlated to the light intensity, with maximum values during night and vice versa. The DF signal showed a strong linear relationship with the quantum yield of photosystem II. Our findings thus suggest that, in addition to the continuous monitoring of active chlorophyll of different taxonomical groups, DF excitation spectrometry also carries the potential to continuously monitor the quantum yields and relative electron transport rates in natural phytoplankton assemblages.