Sulfur-containing Nucleic Acids: Synthesis,Chemical Reactions in Supramolecular Biopolymer Complexes,Biological Applications

The chemical behavior of sulfur-containing oligonucleotides and their reactivity in self-assembled nucleic acids (NA) and specific NA–protein complexes is considered. Reviewed are postsynthetic approaches that allow introducing sulfur-containing linkages at preselected positions of the sugar-phosphate backbone of DNA and between neighboring nucleobases, to incorporate disulfide bridges between complementary strands of double- and triple-stranded DNAs, in large catalytic RNA, etc. Special reference is given to the site-specific chemical modifications as a tool for elucidating the structure, folding, and function of biomolecules. Structure-directed chemical reactions are shown to be helpful in detecting point mutations in DNA, targeting the modifications on specific positions of NA, probing the molecular recognition in protein–DNA interfaces, studying the conformational dynamics of nucleic acids, and discriminating between different folding models.     

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H,¹⁵N,¹³C labelled intact and (des2–7) trp repressor (2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³C positions in both forms of the protein and for¹³ C in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of 2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound 2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and 2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.     

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5"-flap strands of the branched DNA structure and possesses 5"-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3"-end of the primer were synthesized in situ by the action of DNA polymerase using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5"-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5"- and 3"-ends of the nick or with primer–template systems containing 5"- or 3"-protruding DNA strands was shown. Formation of a structure with the 5"-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3"-end and the apurine/apyrimidine site at the 5"-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.