RESUMO
Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.
Assuntos
Compostos Azo/química , Compostos Azo/síntese química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genéticaRESUMO
DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.
Assuntos
Cisteína/metabolismo , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , Compostos Organofosforados/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/química , Metilação de DNA , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC.
Assuntos
Química Orgânica/métodos , Cromatografia Líquida de Alta Pressão , Oligonucleotídeos/química , Aminas/química , Aminoácidos/química , Dióxido de Carbono/química , Ácidos Carboxílicos/química , Cromatografia Líquida de Alta Pressão/métodos , Oligonucleotídeos/síntese química , Peptídeos/químicaRESUMO
DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.
Assuntos
DNA/química , Indicadores e Reagentes/química , Iodoacetatos/química , Ácidos Nucleicos Heteroduplexes/química , Proteínas/química , Nucleotídeos de Uracila/química , Cisteína/química , Dimerização , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , NF-kappa B/química , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Oligonucleotídeos/química , Ribose/químicaRESUMO
The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme-substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A.T by the G.C pair in the methylation site did not affect the enzyme-DNA interaction, whereas the use of a substrate with one chain methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.