The five known RecQ helicases in humans (RECQ1 BLM WRN RECQL4

The five known RecQ helicases in humans (RECQ1 BLM WRN RECQL4 and RECQ5) have demonstrated roles in diverse genome maintenance mechanisms but their functions in safeguarding Rabbit polyclonal to PLCXD1. the genome from environmental toxicants are poorly understood. response. RECQ1-depleted cells exhibited increased RPA phosphorylation Chk1 activation and DNA double strand breaks as compared to control or WRN-depleted cells following exposure AG-014699 to benzo[a]pyrene treatment. Benzo[a]pyrene-induced double strand breaks in RECQ1-depleted cells were dependent on DNA-PK activity. Notably loss of WRN in RECQ1-depleted cells ameliorated benzo[a]pyrene toxicity. Collectively our results provide first indication of nonredundant participation of WRN and RECQ1 in protection from the potentially carcinogenic effects of benzo[a]pyrene and hydroquinone. helicase activity of WRN and RECQ1 proteins was profoundly inhibited by a benzo[a]pyrene diolepoxide adduct with the N2 position of guanosine which is formed upon metabolic activation of BaP in vivo 8 The BaP-DNA adducts are partially resistant to cellular repair processes 9 block replication fork progression and activate the checkpoint kinases ATR and Chk1 in intact cells 10. A very recent study shows that BaP increases double strand break (DSB) repair 11. WRN and RECQ1 are each involved in the repair of stalled and broken replication forks 12-13 although the loss of RECQ1 or WRN contributes differently to the repair of DSB by homologous recombination (HR) 14. Increased HR is also reported in response to oxidative stress induced by benzene metabolites including HQ 15. HQ-induced DNA damage AG-014699 is mediated AG-014699 by the formation of reactive oxygen species 16. Furthermore HQ can be metabolized to potentially genotoxic and carcinogenic benzoquinones that can also induce the formation of free radical predisposing cells to oxidative damage 17. Genetic association studies have previously suggested a link between WRN and susceptibility to benzene-induced toxicity 18. Depletion of WRN is reported to enhance DNA damage in HeLa cells exposed to HQ suggesting that WRN AG-014699 plays a key role in benzene-toxicity 19-20. RECQ1 has also been associated with benzene poisoning 21 but its impact on cell survival following HQ exposure is yet AG-014699 unknown. Deficiency of WRN or RECQ1 results in increased sensitivity to a variety of genotoxic agents accumulation of DNA damage and chromosomal instability associated with cancer predisposition 5 22 Despite the genetic associations of WRN and RECQ1 with cancers 23-25 their roles in governing cellular response to environmental carcinogens have remained poorly characterized. Given the diverse roles of RecQ helicases in nucleic acid metabolism 23 they are likely to be critical in counteracting adverse effects of DNA lesions caused by BaP and HQ. To assess the importance of RECQ1 and WRN in genome surveillance mechanisms activated upon exposure to BaP or HQ we determined survival and DNA damage response to HQ and BaP treatment in RECQ1- and WRN-depleted cells. HeLa cells have been previously used to investigate functions of RecQ helicases 14 26 and mechanisms of HQ 20 or BaP toxicity 4 11 therefore we used HeLa cells as the experimental model. Materials and Methods Chemicals BaP HQ DMSO NU7026 (DNA-PK inhibitor) and KU55933 (ATM inhibitor) were purchased from Sigma. The stock solutions of BaP NU7026 and KU55933 were made in DMSO. HQ stock was made in phosphate-buffered saline (PBS). Cell culture RNA interference and DNA damage treatment HeLa cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories) 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% CO2. Depletion of WRN and RECQ1 was achieved by reverse transfection of WRN or RECQ1 siRNA (10 nM siGenome smartpool Dharmacon) using RNAiMax as per the manufacturer’s instructions (Invitrogen); HeLa cells transfected with scrambled control siRNA were used as control. Cells were treated with various concentrations of HQ or BaP as indicated. Cell proliferation Twenty-four hours after siRNA transfection HeLa cells seeded at a density of 3×103 cells per well in 96 well plates were treated with HQ or BaP in complete DMEM.