Alkylated DNA repair by a novel HhH-GPD family protein from Crenarchaea

HhH-GPD (helix-hairpin-helix-glycine/proline/aspartate) family proteins are involved in DNA damage repair. Currently, mechanism of alkylated DNA repair in Crenarchaea has not been fully clarified. The hyperthermophilic model crenarchaeon Saccharolobus islandicus REY15A possesses a novel HhH-GPD family protein (Sis-HhH-GPD), where its Ser152 corresponds to a conserved catalytic Asp in other HhH-GPD homologs. Herein, we report that Sis-HhH-GPD is a novel bi-functional glycosylase, capable of removing both 1-methyladenine (1-meA) from DNA and alkylated bases from DNA created by methyl methanesulfonate (MMS). Mutational analyses show that E134 is essential for catalysis, whereas S152 is not essential. Sis-HhH-GPD might utilize aromatic rings of Y154 and W57 to stack against 1-meA base for flipping-out and then be removed by E134. Additionally, R157, R161 and R200 participate in catalysis. Among four cysteine residues that potentially coordinate with the Fe-S cluster loop, C203, C210 and C219 are involved in catalysis. Importantly, Sis-HhH-GPD is responsible for repair of alkylated DNA created by MMS in vivo. Interestingly, genetic complementary data have confirmed physiological function of Sis-HhH-GPD in alkylated DNA repair and clarified functional roles of its four cysteine residues in vivo. Overall, we provide first evidence that HhH-GPD family protein from Crenarchaea functions in alkylated DNA repair.