The role of ATR mediated p53 phosphorylation at Serine 15 in Homologous Recombination

Abstract

The purpose of this study was to investigate the influence of ATR- mediated phosphorylation of p53 at S15 on Homologous recombination. Since DNA repair pathways, enzymes and their up- and downregulation play an important role in cancer genesis and moreover attain increasing importance in cancer treatment and therapy, it is crucial to gain further knowledge of the exact molecular mechanisms that promote or suppress the respective repair pathways. In this study, Rad51 foci formation were employed as indicator of recombination activity. HR levels of three mutant cell strains were compared to determine whether ATR-mediated phosphorylation of p53 at S15 results in HR suppression. The cell system used for the experimental layout has been derived from the NSCLC H1299 cell line and consists of three variants of p53 mutants, one p53 null cell line, one p53 L22Q,W23S mutant and transactivation-inactive cell line and one L22Q,W23S S15A mutant cell line, which is transactivation-inactive and harbors an additional deficiency that prevents phosphorylation at S15 via ATR. Changes in HR levels or frequency in all cell lines were measured via quantification of Rad51 foci accumulation. Replicative stress was induced via replication fork stalling through thymidine, which is thought to mainly activate ATR as the principal damage response kinase following replication fork stalling. The inhibition of ATR was investigated via incubation with different concentrations of Caffeine. This study was able to demonstrate that S-15 phosphorylation of p53 via ATR results in significant downregulation of HR. The presented results indicated a clearly impaired HR downregulation in the S15A mutated cell line compared to the other cell lines. Further, the kinetics of the Rad51 foci formation during the course (2-24h) of TdR mediated replication fork stalling, as well as after release (24h +2h,4h,etc), were investigated and described. Despite the known effect of a rise in HR levels in all three cell strains during 24h of TdR treatment, the time course revealed an additional strong increase in HR activity similar in all three cell strains after release from 24h replication fork stalling. In order to study whether DNA damage has been cause of Rad51 accumulation, the formation of γ-H2AX foci during the same time period was studied. It was observed that the Rad51 foci accumulation was accompanied by a simultaneous, though not equally strong rise in levels of γ-H2AX foci, due to several presumably corresponding replication fork restart pathways. The inhibition of ATR/ATM through incubation with different Caffeine concentrations resulted in an initial strong increase in HR levels at 0.4mM Cf, followed by intermediate HR levels at 2mM, and a complete inhibition of HR at 5mM Cf, indicating additional, p53-independent regulatory activities of ATR/ATM upon HR downregulation. Therefore, ATR-dependent phosphorylation of p53 at S-15 could serve as one of several coexisting downregulation mechanisms to suppress excessive HR and to reduce the risk of malignant transformation.