Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Animais , Transformação Celular Neoplásica , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Humanos , Leucemia Mieloide Aguda/genética , Mutagênese , Recombinação Genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Xeroderma Pigmentoso/genéticaRESUMO
Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.
Assuntos
Desoxirribodipirimidina Fotoliase/metabolismo , Flavinas/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Quimotripsina , Clonagem Molecular , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/isolamento & purificação , Escherichia coli/genética , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Mapeamento por Restrição , Riboflavina/análogos & derivados , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
The nucleotide sequence of a 2301 base pair region of Saccharomyces cerevisiae DNA containing the PHR1 gene is reported. Within this region a single open reading frame of 1695 base pairs was found; using the insertional inactivation technique it was shown that part or all of this open reading frame specifies the PHR1-encoded photolyase. The amino acid sequence of the 565 amino acid long polypeptide predicted from the PHR1 nucleotide sequence was compared to the amino acid sequence of E. coli photolyase. Overall the sequence homology was 36.5%; however, two short regions near the amino terminus as well as the carboxy-terminal 150 amino acids display significantly greater sequence homology. The presence of these strongly conserved regions suggests that the yeast and E. coli photolyase possess common structural and functional domains involved in substrate and/or chromophore binding.
Assuntos
Desoxirribodipirimidina Fotoliase/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Liases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Códon , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
We have determined the sequence of a 2400 bp region of E. coli chromosomal DNA containing the uvrC gene. The coding region of uvrc is 2267 bp in length, encodes a polypeptide with a calculated molecular weight of 66,038 daltons, and is preceded by a typical E. coli ribosome binding site. By constructing deletion derivatives we have established that a uvrC promoter lies within the 113 bp region preceding the translational start of uvrC. The codon usage in uvrC is strongly biased in favor of codons used infrequently in E. coli, which may contribute to the relatively low intracellular concentration of uvrC protein.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Códon/genética , Enzimas de Restrição do DNA , Escherichia coli/efeitos da radiação , Peso Molecular , Óperon , Plasmídeos , Biossíntese de Proteínas , Raios UltravioletaRESUMO
Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.