The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach1

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.

Mitochondrial Single-stranded DNA-binding Protein Is Required for Mitochondrial DNA Replication and Development in Drosophila melanogaster

The discovery that several inherited human diseases are caused by mtDNA depletion has led to an increased interest in the replication and maintenance of mtDNA. We have isolated a new mutant in the lopo (low power) gene from Drosophila melanogaster affecting the mitochondrial single-stranded DNA-binding protein (mtSSB), which is one of the key components in mtDNA replication and maintenance. lopo1 mutants die late in the third instar before completion of metamorphosis because of a failure in cell proliferation. Molecular, histochemical, and physiological experiments show a drastic decrease in mtDNA content that is coupled with the loss of respiration in these mutants. However, the number and morphology of mitochondria are not greatly affected. Immunocytochemical analysis shows that mtSSB is expressed in all tissues but is highly enriched in proliferating tissues and in the developing oocyte. lopo1 is the first mtSSB mutant in higher eukaryotes, and its analysis demonstrates the essential function of this gene in development, providing an excellent model to study mitochondrial biogenesis in animals.

Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Non-Arrhenius kinetics for the loop closure of a DNA hairpin

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (β = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. & Schulten, Z. (1980) J. Chem. Phys. 72, 4350–4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.

Kinetics and thermodynamics of T cell receptor– autoantigen interactions in murine experimental autoimmune encephalomyelitis

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)–peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1–11) bound to the MHC class II protein, I-Au, and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1–11:I-Au with a 4–5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR–pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

A statistical mechanical model for β-hairpin kinetics

Understanding the mechanism of protein secondary structure formation is an essential part of the protein-folding puzzle. Here, we describe a simple statistical mechanical model for the formation of a β-hairpin, the minimal structural element of the antiparallel β-pleated sheet. The model accurately describes the thermodynamic and kinetic behavior of a 16-residue, β-hairpin-forming peptide, successfully explaining its two-state behavior and apparent negative activation energy for folding. The model classifies structures according to their backbone conformation, defined by 15 pairs of dihedral angles, and is further simplified by considering only the 120 structures with contiguous stretches of native pairs of backbone dihedral angles. This single sequence approximation is tested by comparison with a more complete model that includes the 215 possible conformations and 15 × 215 possible kinetic transitions. Finally, we use the model to predict the equilibrium unfolding curves and kinetics for several variants of the β-hairpin peptide.

UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.