Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomonas aeruginosa MutS by preventing protein oligomerization.
MutS, a component of the mismatch repair system begins the DNA reparation process by recognizing base/base mismatches or small insertion/deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa and analysed the biochemical properties of the encoded protein. Complementation of the hypermutator phenotype of a P. aeruginosa mutS mutant strain indicated that the isolated gene was functional. When purified MutS was incubated at 37 degrees C in the absence of ligands, a rapid inactivation of the oligonucleotide binding capability and ATPase activity occurred. However, the presence of ATP, ADP or heteroduplex oligonucleotides, but not homoduplex oligonucleotides, prevented the protein from being inactivated. The analysis of the protein by native PAGE indicated that the active conformation state correlates with the presence of MutS dimer. Analysis by gel-filtration chromatography showed that the inactive protein formed by incubation at 37 degrees C in the absence of ligands corresponds to the formation of a high molecular mass oligomer. The kinetic analysis of the oligomer formation showed that the extent of the reaction was markedly dependent on the temperature and the presence of MutS ligands. However, the protein inactivation apparently occurred before the maximum extent of MutS oligomerization. Further analysis of the MutS oligomers by electron microscopy showed the presence of regular structures consisting of four subunits, with each subunit probably representing a MutS homodimer. It is concluded that MutS possesses an intrinsic propensity to form oligomeric structures and that the presence of physiological ligands, such as nucleotides or heteroduplex DNA, but not homoduplex DNA, plays an important role in keeping the protein in an active conformation by preventing protein oligomerization.
Pezza
Roberto J
RJ
Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina.
Smania
Andrea M
AM
Barra
José L
JL
Argaraña
Carlos E
CE
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006801:Humans; D008024:Ligands; D008854:Microscopy, Electron; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli