Abstract
The DNA molecule is constantly challenged by endogenous and exogenous agents, threatening the genetic stability. If the damage are not repaired, mutations might accumulate, and possible consequences are cancer, aging and neurodegenerative disease. To preserve the DNAs integrity, a number of repair pathways have evolved. Deamination of bases in DNA can arise spontaneously, after chemical exposure or by enzymatic processes. Deamination of adenine leads to hypoxanthine which is miscoding and potentially mutagenic. Endonuclease V (EndoV) is identified as the main enzyme for initiation of hypoxanthine repair in prokaryotes. EndoV is highly conserved, suggesting an important function for the enzyme.
The eukaryotic homologous of EndoV are poorly characterized and we aimed to identify substrates and function for human EndoV (hEndoV). We characterized hEndoV by bioinformatics, gene expression and in vitro analyses. We found hEndoV to have affinity for various branched DNA substrates. However, we did not find any activity or affinity for hypoxanthine in DNA.
Adenines in RNA are also subjected to deamination and inosine is the most common editing event in RNA (A-to-I editing). Both tRNAs, mRNAs, microRNAs and Alu-containing transcripts are deaminated. Many A-to-I deamination targets are found in the central nervous system and proper editing is important for correct protein function. We found hEndoV to cleave RNA with inosine, a so far undescribed activity.
The structure of Thermotoga maritima EndoV (TmEndoV) has been solved in complex with DNA with hypoxanthine. The structure revealed a conserved wedge motif which separates the two DNA strands. To further investigate the role of the wedge, the crystal structure of TmEndoV in complex with a one-nucleotide loop was solved. The structure shows how the wedge separates the two DNA strands at the helical distortion and a normal adenine is flipped into the recognition pocket.