Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomona aeruginosa MutS by preventing protein oligomerization.

PEZZA R,; SMANIA A.M.,; JOSÉ LUIS BARRA; ARGARAÑA C.E,

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 2002 vol. 361 p. 87 - 95

0264-6021

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 from the human opportunistic pathogen Pseudomonas aeruginosa or small insertion}deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa from the human opportunistic pathogen Pseudomonas aeruginosa }base mismatches or small insertion}deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa from the human opportunistic pathogen Pseudomonas aeruginosa }deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosaPseudomonas 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 puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high mutS mutant strain indicated that the isolated gene was functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high P. aeruginosa mutS mutant strain indicated that the isolated gene was functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high mutant strain indicated that the isolated gene was functional. When puri®ed MutS was incubated at 37 °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high 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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high °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-®ltration chromatography showed that the inactive protein formed by incubation at 37 °C in the absence of ligands corresponds to the formation of a high absence of ligands corresponds to the formation of a high °C in the absence of ligands corresponds to the formation of a high INTRODUCTION The MutS protein is part of the MutSLH mismatch repair system (MMRS) which prevents genome alterations in prokaryotes and eukaryotes. This system avoids point mutations or small insertions}} deletions by correcting replication errors, as well as chromosomal rearrangements by preventing recombination between homologous sequences [1,2]. In Escherichia coli, the assembly of the MMRS to repair DNA biosynthetic errors is initiated by MutS, which recognizes and binds to mispaired nucleotides. Bound MutS interacts with MutL, which is supposed to promote the translocation of the complex to a hemi-methylated d(GATC) site where MutH is bound. This interaction stimulates an endonuclease activity of MutH, which produces a nick in the newly synthesized unmethylated DNA strand. The excision of the nascent strand is catalysed by a single-strand DNA-speci®c exonuclease. Finally, DNA polymerase III holoenzyme and DNA ligase carry out the resynthesis and ligation steps to restore the correct complementary sequence [1±3]. A weak ATPase activity is an evolutionary conserved characteristic of MutS [4±8]. This activity appears to be relevant for its physiological function, since mutations of the conserved nucleotide- binding domain result in a dominant mutator phenotype in both bacteria and yeast [4,9,10]. Besides the ATPase activity, the binding of adenine nucleotides to MutS plays an important role in the functioning of the MMRS. It has been shown that the presence of ATP reduces the affinity of MutS for the heteroduplex and it was suggested that the ATP hydrolysis energy is used for translocation of the repair complex towards the hemi-methylated biosynthetic errors is initiated by MutS, which recognizes and binds to mispaired nucleotides. Bound MutS interacts with MutL, which is supposed to promote the translocation of the complex to a hemi-methylated d(GATC) site where MutH is bound. This interaction stimulates an endonuclease activity of MutH, which produces a nick in the newly synthesized unmethylated DNA strand. The excision of the nascent strand is catalysed by a single-strand DNA-speci®c exonuclease. Finally, DNA polymerase III holoenzyme and DNA ligase carry out the resynthesis and ligation steps to restore the correct complementary sequence [1±3]. A weak ATPase activity is an evolutionary conserved characteristic of MutS [4±8]. This activity appears to be relevant for its physiological function, since mutations of the conserved nucleotide- binding domain result in a dominant mutator phenotype in both bacteria and yeast [4,9,10]. Besides the ATPase activity, the binding of adenine nucleotides to MutS plays an important role in the functioning of the MMRS. It has been shown that the presence of ATP reduces the affinity of MutS for the heteroduplex and it was suggested that the ATP hydrolysis energy is used for translocation of the repair complex towards the hemi-methylated Escherichia coli, the assembly of the MMRS to repair DNA biosynthetic errors is initiated by MutS, which recognizes and binds to mispaired nucleotides. Bound MutS interacts with MutL, which is supposed to promote the translocation of the complex to a hemi-methylated d(GATC) site where MutH is bound. This interaction stimulates an endonuclease activity of MutH, which produces a nick in the newly synthesized unmethylated DNA strand. The excision of the nascent strand is catalysed by a single-strand DNA-speci®c exonuclease. Finally, DNA polymerase III holoenzyme and DNA ligase carry out the resynthesis and ligation steps to restore the correct complementary sequence [1±3]. A weak ATPase activity is an evolutionary conserved characteristic of MutS [4±8]. This activity appears to be relevant for its physiological function, since mutations of the conserved nucleotide- binding domain result in a dominant mutator phenotype in both bacteria and yeast [4,9,10]. Besides the ATPase activity, the binding of adenine nucleotides to MutS plays an important role in the functioning of the MMRS. It has been shown that the presence of ATP reduces the affinity of MutS for the heteroduplex and it was suggested that the ATP hydrolysis energy is used for translocation of the repair complex towards the hemi-methylated Abbreviations used: DTT, dithiothreitol ; IPTG, isopropyl b-D-thiogalactoside; LB, Luria±Bertani; MMRS, mismatch repair system.b-D-thiogalactoside; LB, Luria±Bertani; MMRS, mismatch repair system. 1 To whom correspondence should be addressed (e-mail carga!dqb.fcq.unc.edu.ar).To whom correspondence should be addressed (e-mail carga!dqb.fcq.unc.edu.ar). 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.