Many biological processes undergo daily (circadian) rhythms that are dictated by self-sustained biochemical oscillators. These circadian clock systems generate a ~24 h period in constant conditions (i.e. light and temperature) that is nearly invariant at different temperatures. Circadian clocks also show entrainment to day and night, predominantly mediated by the daily light/dark cycle, so that the endogenous biological clock is phased appropriately to the environmental cycle. A full understanding of these unusual oscillators will require knowledge of the structures, functions, and interactions of their molecular components. We are studying the components of the biological clock in the prokaryotic cyanobacterium Synechococcus elongatus that programs many processes to conform optimally to the daily cycle, including photosynthesis and nitrogen fixation. The endogenous circadian system in cyanobacteria also exerts pervasive control over cellular processes including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topological changes and compaction. Remarkably, the biochemical machinery underlying this circadian oscillator can be reconstituted in vitro with just three cyanobacterial proteins, KaiA, KaiB, and KaiC in the presence of ATP! These proteins interact to promote conformational changes and phosphorylation events that determine the phase of the in vitro oscillation. The KaiC homo-hexamer forms the central cog of the clock and is an auto-kinase and -phosphatase and an ATPase. The KaiA dimer enhances KaiC phosphorylation and KaiB dimers antagonize KaiA's action. The high-resolution structures of these proteins suggest a racheting mechanism by which the KaiABC oscillator ticks unidirectionally. We are dissecting the mechanism of the KaiABC clock using hybrid structural techniques, including X-ray crystallography and electron microscopy as well as a range of biophysical and biochemical approaches and assays in vivo and in vitro. [Supported by NIH grant R01 GM073845]

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The human MutS homologs (MSH), hMSH2 and hMSH6, forms a mutually dependent heterodimer (hMSH2-hMSH6). The hMSH2-hMSH6 protein plays a central role in mismatch repair (MMR). hMSH2-hMSH6 is required for the recognition of mismatched nucleotides generated by misincorporation during DNA replication thereby maintaining genomic stability. Mutations in the hMSH2 or hMSH6 genes result in elevated spontaneous mutation rate and susceptibility to the common cancer predisposition syndrome, Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

DNA repair in vivo is complicated by the fact that mismatches/lesions arise within chromosomes that are a complex mixture of DNA and protein (chromatin). A fundamental unit of chromatin is a nucleosome which consists of ∼146 bp of DNA wrapped two times around a histone octomer of two H2A-H2B dimers and an H3-H4 tetramer. The biophysical effect of MMR on chromatin is unknown. Moreover, little is known about the effect of the more than 100 post-translational modifications that may decorate the human histones on MMR processes.

We have developed a mismatched DNA substrate containing a single well-defined nucleosome. The nucleosome was reconstituted from histones overproduced and purified from bacteria. We demonstrate that hMSH2-hMSH6 can catalyze the displacement of a nucleosome adjacent to a mismatch. Displacement of the nucleosome requires ATP binding by hMSH2-hMSH6. Nucleosome displacement is blocked by LacI/LacO placed between the mismatch and the nucleosome arguing in favor of a "cis" or "moving" mechanism. In addition, we have reconstructed nucleosomes containing specific modifications of histone H3 by a semi-synthetic intein-based strategy. We find that hMSH2-hMSH6 nucleosome displacement is considerably enhanced when histone H3 is acetylated at the dyad residues K115 and K122. H3(K115, K122) acetylation enhances the intrinsic mobility of the nucleosome on DNA in vitro and occurs during replication and repair in vivo. These results highlight a number of unanticipated strengths of the Sliding Clamp Model for MMR and identify a "Regulated Nucleosome Mobility Model" for chromatin remodeling during DNA repair.

Mitochondria have long been known to sequester cytosolic Ca and even to shape intracellular patterns of endoplasmic reticulum-based Ca signaling. Evidence suggests that the mitochondrial network is an excitable medium which can demonstrate independent Ca induced Ca release via the mitochondrial permeability transition. The role of this excitability remains unclear, but mitochondrial Ca handling appears to be a crucial element in diverse diseases as diabetes, neurodegeneration and cardiac dysfunction that also have bioenergetic components. In this paper, we extend the modular Magnus-Keizer computational model for respiration-driven Ca handling to include a permeability transition based on a channel-like pore mechanism. We demonstrate both excitability and Ca wave propagation accompanied by depolarizations similar to those reported in cell and isolated mitochondria preparations. These waves depend on the energy state of the mitochondria, as well as other elements of mitochondrial physiology. Our results support the concept that mitochondria can transmit state dependent signals about their function in a spatially extended manner.