The dichotomous effects of caffeine on homologous recombination in mammalian cells

Abstract

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR). An initial 2 h exposure to 5 mM caffeine slowed a fraction of the cells in G1, but thereafter, continued caffeine exposure permitted this cell fraction to progress through the cycle until they eventually stalled at G2/M and underwent apoptosis. This prolonged caffeine exposure also induced a strong DDR along with subsequent activation of wild-type p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Conversely, the increase in nascent DNA synthesis did not translate into a higher level of gene targeting events. Levels of Rad51 appear to be irrelevant. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and stimulates early steps of HR, but not the formation of complete repair products. INTRODUCTIONThe DNA damage response (DDR) is a complex signalling pathway consisting of sensors, transducers and effectors that evolved to detect DNA damage, halt the cell cycle and co-ordinate an adaptive response (1). In mammalian cells, the protein kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM-and Rad3-related), both members of the phosphatidylinositol 3kinase-related kinase (PIKK) superfamily of protein kinases play a key role in DDR signalling (2). The CHK1 and CHK2 protein kinases are two of the best studied ATM/ATR targets and in association with ATM and ATR, reduce cyclin-dependent kinase (CDK) activity by mechanisms that include activation of the p53 transcription factor (3). CDK inhibition reduces or halts cell cycle progression at either of the G1-S, intra-S or G2-M cell cycle checkpoint boundaries (3). Cell cycle arrest normally permits repair of DNA damage prior to DNA replication or mitosis through ATM-and ATR-catalyzed induction of DNA repair protein transcription or posttranscriptional activation, recruitment of DNA repair factors to the damaged site and/or their