The role of MutS oligomers on Pseudomonas aeruginosa Mismatch Repair System activity

MONTI MARIELA R.*; MIGUEL VIRGINIA*; ARGARAÑA CARLOS E.; CO-AUTORAS

DNA REPAIR

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2008 vol. 7 p. 1799 - 1808

1568-7864

The Escherichia coli DNA Mismatch Repair (MMR) protein MutS exist as dimers and tetramers in solution, and the identification of its functional oligomeric state has been matter of extensive study. In the present work, we have analyzed the oligomerization state of MutS from Pseudomonas aeruginosa a bacterial species devoid of Dam methylation and MutH homologue. By analyzing native MutS and different mutated versions of the protein, we determined that P. aeruginosa MutS is mainly tetrameric in solution and that its oligomerization capacity is conducted as in E. coli, by the C-terminal region of the protein. The analysis of mismatch oligonucleotide binding activity showed that wild-type MutS binds to DNA as tetramer. The DNA binding activity decreased when the C-terminal region was deleted (MutS <!-- /* Font Definitions */ @font-face {font-family:"Palatino Linotype"; panose-1:2 4 5 2 5 5 5 3 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870009 1073741843 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Ä798) or when a full-length MutS with tetramerization defects (MutSR842E) was tested. The ATPase activity of MutS <!-- /* Font Definitions */ @font-face {font-family:"Palatino Linotype"; panose-1:2 4 5 2 5 5 5 3 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870009 1073741843 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Ä798 was similar to MutSR842E and diminished respect to the wild-type protein. Experiments carried out on a P. aeruginosa mutS strain to test the proficiency of different oligomeric versions of MutS to function in vivo showed that MutS <!-- /* Font Definitions */ @font-face {font-family:"Palatino Linotype"; panose-1:2 4 5 2 5 5 5 3 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870009 1073741843 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Ä798 is not functional and that full-length dimeric version MutSR842E, is not capable of completely restoring the MMR activity of the mutant strain. Additional experiments carried out in conditions of high mutation rate induced by the base analogue 2-AP confirm that the dimeric version of MutS is not as efficient as the tetrameric wild-type protein to prevent mutations. Therefore, it is concluded that although dimeric MutS is sufficient for MMR activity, optimal activity is obtained with the tetrameric version of the protein and therefore it should be considered as the active form of MutS in P. aeruginosa.