RESUMEN
To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.
Asunto(s)
Adenina/análogos & derivados , Proteínas Bacterianas/fisiología , ADN Glicosilasas , Reparación del ADN , Proteínas de Escherichia coli , Escherichia coli/enzimología , Guanina/análogos & derivados , N-Glicosil Hidrolasas/fisiología , Adenina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Disparidad de Par Base , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/fisiología , Catálisis , Dominio Catalítico , Daño del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/genética , Guanina/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/química , Monoéster Fosfórico Hidrolasas/química , Conformación Proteica , Estructura Terciaria de Proteína , Pirofosfatasas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
One of the functions of MutY from Escherchia coli is removal of adenine mispaired with 7,8-dihydro-8-oxoguanine (8-oxoG), a common lesion in oxidatively damaged DNA. MutY is composed of two domains: the larger N-terminal domain (p26) contains the catalytic properties of the enzyme while the C-terminal domain (p13) affects substrate recognition and enzyme turnover. On the basis of sequence analyses, it has been recently suggested that the C-terminal domain is distantly related to MutT, a dNTPase which hydrolyzes 8-oxo-dGTP [Noll et al. (1999) Biochemistry 38, 6374-6379]. We have studied the solution structure of the C-terminal domain of MutY by NMR and find striking similarity with the reported solution structure of MutT. Despite low sequence identity between the two proteins, they have similar secondary structure and topology. The C-terminal domain of MutY is composed of two alpha-helices and five beta-strands. The NOESY data indicate that the protein has two beta-sheets. MutT is also a mixed alpha/beta protein with two helices and two beta-sheets composed of five strands. The secondary structure elements are similarly arranged in the two proteins.
