BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
The reaction of 3,4-epoxy-1-butene (BMO) with deoxyguanosine-3'-monophosphate (3'-dGMP) resulted in the formation of two pairs of diastereomeric 7-alkyl-3'-dGMP derivatives corresponding to two isomers C?-1 and C?-2. The T4 polynucleotide kinase-mediated phosphorylation with [gamma-32P]-ATP showed preferential labelling of diastereo- mers of the C?-1 isomer. The diastereomers 1 and 2 of the C?-1 isomer had labelling efficiencies of 42%. However, the labelling efficiencies of diastereomers 3 and 4 of the C?-2 isomer were 11 and 10%, respectively. The 32P-postlabelling of BMO-modified DNA yielded four isomers in the ratio of 4:4:1:1 with overall recoveries being 14%. The two isomers had a half-life of 270 min (C?-1 isomer) and 300 min (C?-2 isomer) which is in accordance with the stability predicted by other similar adduct experiments. The molecular modelling experiments showed more pronounced restricted rotation of butadiene residue in C?-2 isomers due to steric interaction between butadiene residue at N-7 and O(6) atom of guanine than in C?-1 isomer. The butadiene residue also leads to steric overcrowding at 3'-phosphate in C?-2 isomer which probably restricts the access to the active site of T4 polynucleotide kinase.
- MeSH
- adukty DNA analýza biosyntéza izolace a purifikace MeSH
- deoxyguaninnukleotidy izolace a purifikace metabolismus MeSH
- DNA metabolismus účinky léků MeSH
- epoxidové sloučeniny farmakologie metabolismus MeSH
- fosforylace MeSH
- izotopové značení MeSH
- losos MeSH
- molekulární modely MeSH
- mutageny farmakologie izolace a purifikace metabolismus MeSH
- poločas MeSH
- polynukleotid-5'-hydroxylkinasa metabolismus MeSH
- radioizotopy fosforu MeSH
- stereoizomerie MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
