potassium-permanganate and hydrazine

The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an initiation complex for DNA unwinding. We studied the structure of these complexes formed with blunt-ended, 5'-extended, and 3'-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg(2+)-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5' end were only partially reactive to KMnO4, suggesting that part of the 5'-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.

Topics: Adenosine Triphosphate; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Helicases; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Hydrazines; Hydroxyl Radical; Magnesium; Models, Chemical; Nucleic Acid Conformation; Potassium Permanganate; Sulfuric Acid Esters

Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats. RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo. We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting. Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY. Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides. In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex. The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp. This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes.

Topics: Amino Acid Sequence; Base Sequence; Chromosome Mapping; Dimethyl Sulfoxide; DNA-Binding Proteins; DNA, Fungal; Hydrazines; Molecular Sequence Data; Nucleic Acid Conformation; Potassium Permanganate; Regulatory Sequences, Nucleic Acid; Saccharomyces cerevisiae; Telomere; Transcription Factors