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The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

The transcription of genes located in subtelomeric regions of yeast chromosomes is repressed relative to the rest of the genome. This repression requires wild-type nucleosome levels but not the telomere silencing factors Sir2, Sir3, Sir4, and Rap1. Subtelomeric heterochromatin is characterized by the absence of acetylation or methylation of histone H3 lysine residues, but it is not known whether histone H3 hypoacetylation or hypomethylation is a prerequisite for the establishment of subtelomeric heterochromatin. We have systematically mutated the N-terminal tails of histone H3 and H4 in Saccharomyces cerevisiae and characterized the effects each mutant has on genome-wide expression. Our results show that subtelomeric transcriptional repression is dependent on the histone H3 N-terminal domain, but not the histone H4 N-terminal domain. Mutating lysine4, lysine-9, lysine-14, lysine-18, lysine-23, and lysine-27 to glycine in histone H3 is also sufficient to significantly reduce subtelomeric gene repression. Individual histone H3 lysine mutations, however, have little effect on subtelomeric gene repression or genome-wide expression, indicating that these six lysine residues have redundant functions. We propose that acetylation and methylation of histone H3 N-terminal lysine residues act as redundant mechanisms to demarcate regions of euchromatin from heterochromatin.

Results show that the conformational changes that mediate the active site assembly and metal binding by EcoRV endonuclease are modulated by the electrostatic effects in the DNA cleavage region of the enzyme. Kinetic and crystallographic analyses of the charged active site mutants reveal that site-specific mutations do not affect enzyme activity.

Analysis of DNA exposed to UV radiations at 254nm and 100 to 10,000 J m2 reveals the presence of three monomeric products, of which two are formamidopyrimidine derivatives, and the third is a pyrimidine derivative. These monomers act as substrates for base excision enzymes that repair oxidatively damaged DNA. The yield of the thymine derivative depends directly on intensity of UV radiation, while the quantity of the other derivatives obtained vary nonlinearly. Increased dosage of radiations also induces the formation of several other derivatives of purine and pyrimidine.

The combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measures, the complete catalytic cycle of EcoRV endonuclease. Binding, bending and cleavage of substrate oligonucleotide were monitored in real time anisotropy.

Research was conducted to examine the divalent metal dependence of site-specific DNA binding by EcoRV endonuclease. For short duplex oligodeoxynucleotides having the cognate DNA site, three cleavable noncognate sites and a fully nonspecific site, the binding constants were determined. Results provide strong evidence that measurements of DNA binding affinities for EcoRV should be conducted in the presence of divalent metal ions.

Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups. Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol?1 toward specificity, with an additional 6?10 kcal mol?1 arising from indirect readout. Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA. [ABSTRACT FROM AUTHOR]