Arginine methylation is a post-translational modification occurring in higher eukaryotes that results in the addition of one or two methyl group on the nitrogen in the side chain of arginines. The enzymes responsible for protein arginine methylation have been classified in three groups. Type I enzymes promote the formation of both NG-monomethylated and asymmetric o-NG,NG-dimethylated arginines (aDMA). Type II enzymes catalyze the formation of monomethylated and symmetrical o-N G,N'G-dimethylated arginines (sDMA). The type III enzyme found in yeast catalyzes the monomethylation of the delta-guanidino nitrogen atom of the arginine residue. Although some abundant proteins have been described as being substrates for arginine methyltransferases for some time, there are still few known proteins to bear this modification. The primary goal of the work presented in this thesis was to identify new arginine methylated proteins and functionally characterize the roles of arginine methylation in new cellular processes. First, we generated four arginine methyl-specific antibodies: ASYM24 and ASYM25 are specific for aDMA whereas SYM10 and SYM11 recognize sDMA. Cell extracts were used to purify the protein complexes recognized by each of the four antibodies and the proteins were identified by microcapillary reverse-phase liquid chromatography coupled on line with electrospray ionization tandem mass spectrometry (LC/MS/MS). The analysis of 2 tandem mass spectra for each methyl-specific antibody resulted in the identification of 247 proteins, of which 197 are putatively arginine methylated. / The DNA repair MRE11/RAD50/NBS1 (MRN) complex was purified using one of the aDMA specific antibody. Since a role of protein arginine methylation in DNA damage checkpoint control and DNA repair had not been previously reported we chose to investigate the consequence of MRE11 methylation in DNA damage. Our results show that the MRE11 checkpoint protein is arginine methylated as determined by mass spectrometry and methylarginine-specific antibodies. The glycine-arginine rich (GAR) domain of MRE11 was specifically methylated by protein arginine methyltransferase 1 (PRMT1). Mutation of the arginines within MRE11 GAR domain severely impaired the exonuclease activity of MRE11. Cells treated with methyltransferase inhibitors displayed a DNA damage-resistant DNA synthesis phenotype and prevented the re-localization of the MRN complex to sites of DNA damage. Downregulation of PRMT1 with small interfering RNAs (siRNA) also yielded a damage-resistant DNA synthesis phenotype that was rescued with the methylated MRE11 complex. Taken together, the work presented in this thesis allowed the identification of many new potentially arginine methylated proteins and demonstrated a novel role for arginine methylation in the regulation of DNA repair enzymes and of the intra-S phase DNA damage checkpoint.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85887 |
| Date | January 2005 |
| Creators | Boisvert, François-Michel |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002261178, proquestno: AAINR21624, Theses scanned by UMI/ProQuest. |
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