Functional Studies Of A-family DNA Polymerases In The Response To Oxidative DNA Damage In Human Cells

Both exogenous environmental factors and endogenous intracellular metabolites can induce oxidative DNA damage.8-oxo-7,8-dihydroguanine(8-oxoG)is one of the most common oxidative DNA lesions,which is considered to be a crucial biomarker of DNA oxidative damage.Once oxidative DNA damage occurs,cells will initiate DNA damage response mechanisms,including DNA damage repair and translesion DNA synthesis(TLS).The A-family polymerases,Polymerase ν(Pol ν)and Polymerase ν(Pol θ),are novel kinds of TLS polymerases.However,their biological functions in the oxidative DNA damage response in mammalian cells remain largely unknown.Here,we first assessed the potential functions of Pol ν and Pol θ during DNA oxidative stress through cell survival assays.In addition,we employed a robust shuttle vector method combined with next-generation sequencing to explore how 8-oxoG affects the transmission of genetic information in HEK293T cells with or without the knockdown of Pol ν-encoding gene(POLN)or Pol θ-encoding gene(POLQ).The main research contents are as follows:(1)Investigation of the potential roles of A-family DNA polymerases in oxidative stressinduced cellular damage response.First,we achieved the efficient knockdown of POLN or POLQ in human cells by short hairpin RNA(shRNA).Based on the H2O2-induced oxidative stress model,we initially explored the biological functions of Pol ν or Pol θ in oxidative stress.The results showed that the knock-down of POLN or POLQ cells exhibited higher sensitivity to oxidative DNA damage-induced cytotoxicity.Therefore,these results suggested that Pol νor Pol θ may be involved in oxidative DNA damage response in human cells.(2)Assessment of the potential roles of A-family DNA polymerases in the replicative bypass of 8-oxoG in vivo.First,we examined the efficiencies and accuracies of replicative bypass of the 8-oxoG lesion by utilizing a shuttle-vector-based method combined with nextgeneration sequencing analysis.The results showed that 8-oxoG lesion apparently blocked the DNA replication and induced G→T transversion in human cells.To further explore the biological functions of Pol ν or Pol θ in oxidative DNA damage response,we also investigated how the effects of 8-oxoG on DNA replication are influenced by the knock-down of POLN or POLQ in HEK293T cells.We observed significant diminutions in bypass efficiencies for 8oxoG lesion in POLN or POLQ knockdown cells relative to the wild-type cells.These results implied that Pol ν and Pol θ are likely to play an important role in TLS across the 8-oxoG.(3)Examination of the potential roles of A-family DNA polymerases in the transcriptional bypass of 8-oxoG in vivo.Considering that specialized TLS polymerases have been reported to play important roles in the transcriptional bypass of alkylated DNA damage,we further investigated the potential involvement of Pol ν or Pol θ in the transcriptional process 8-oxoG in cells.We employed the shuttle vector and next-generation sequencing-based strategy to explore the efficiencies and fidelities of DNA transcription across 8-oxoG in POLN or POLQ knockdown cells.The results indicated that the knock-down of POLN or POLQ did not significantly perturb the bypass efficiencies and mutation frequencies of transcription across 8oxoG,implying that Pol ν and Pol θ may not be involved in the transcriptional process of this DNA oxidative damage.In conclusion,we have systematically investigated the potential functions of A-family DNA polymerases in the oxidative DNA damage response.Our results showed that Pol ν and Pol θ,to some extent,enhanced cellular tolerance to oxidative agents and reduced oxidative damage-induced cytotoxicity,and revealed the critical roles of Pol ν and Pol θ in promoting the replicative bypass of 8-oxoG in human cells.These findings would promote a better understanding of the biological functions of A-family DNA polymerases and the molecular mechanisms of oxidative DNA damage in human diseases.