First evidence of in vivo DNA glycosylase activity of the Arabidopsis AtMBD4L enzyme

TORRES JR; LESCANO CI; ALVAREZ ME

Virtual

Congreso; Congreso Conjunto SAIB-SAMIGE 2020; 2020

SAIB-SAMIGE

DNA glycosylases catalyze the first step of the Base Excision Repair (BER) pathway. MBD4 is a monofunctional DNA glycosylase from the Helix-hairpin-Helix GDP (HhH-GDP) superfamily that recognizes Uracil (U) or Thymine (T) opposite at Guanine (G), as well as the presence of halogenated derivatives of U such as 5-Bromouracil (5-BrU) and 5-Fluorouracil (5-FU), in DNA. The Arabidopsis MBD4 homologue, called AtMBD4L, is being characterized at both genic and enzymatic levels. In vitro, recombinant AtMBD4L recognizes and excises 5-BrU, 5-FU and U from synthetic oligonucleotides with high efficiency. In vivo the AtMBD4L protein shows nuclear localization and has two splicing isoforms. However, the roles of AtMBD4L on the BER pathway in vivo have not been determined yet. We here analyze the participation of AtMBD4L in recognizing and exercising U and 5-FU from the Arabidopsis nuclear genome in vivo. For that, we exposed wild type (WT) and AtMBD4L mutant (mbd4l) plants to different concentrations of 5-FU, and analyzed different phenotypes, such as seed germination, seedling and principal root growth, root meristematic cell death and root tip morphology. In addition, we assessed DNA damage by using comet assays. Seed germination, and seedling and principal root growth were lower in mbd4l than WT plants after 5-FU treatment. In contrast, the number death cells in the root meristematic zone was dramatically higher in mbd4l than WT plants. In addition, the root tip morphology was highly affected in mbd4l compared to WT plants. Lastly, the DNA breaks were significantly higher in WT than mbd4l plants under this condition. Together these results provide the first evidence for the role of AtMBD4L in vivo, suggesting that this enzyme is required for maintenance of genome integrity and plant growth in the presence of 5-FU.