title: Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells.
abstract: DNA polymerase eta (Pol η) is one of several Y family trans-lesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased both with empty vector control (EVC), wild-type POLH, and Y52E-POLH over 48 hours post UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV induced DNA lesions in HaCaT cells.
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: C
authors.initials: C
authors.lastname: Luo
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: Q
authors.initials: Q
authors.lastname: He
authors.affiliation: Department of Oncology, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China. caokehui168@aliyun.com.
authors.forename: K
authors.initials: K
authors.lastname: Cao
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.
authors.forename: R
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Burn and Plastic, Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China. doctorzhoujianda@163.com.
authors.forename: J
authors.initials: J
authors.lastname: Zhou