Abstract
Cytosine deamination into uracil is one of the most prevalent and pro-mutagenic forms of damage to DNA. Base excision repair is a well-known process of uracil removal in DNA, which is achieved by uracil DNA glycosylase (UDG) that is found in all three domains of life. However, other strategies for uracil removal seem to have been evolved in Archaea. Exonuclease III (ExoIII) from the euryarchaeon Methanothermobacter thermautotrophicus has been described to exhibit endonuclease activity toward uracil-containing DNA. Another uracil-acting protein, endonuclease Q (EndoQ), was recently identified from the euryarchaeon Pyrococcus furiosus. Here, we describe the uracil-counteracting system in the mesophilic euryarchaeon Methanosarcina acetivorans through genomic sequence analyses and biochemical characterizations. Three enzymes, UDG, ExoIII, and EndoQ, from M. acetivorans exhibited uracil cleavage activities in DNA with a distinct range of substrate specificities in vitro, and the transcripts for these three enzymes were detected in the M. acetivorans cells. Thus, this organism appears to conduct uracil repair using at least three distinct pathways. Distribution of the homologs of these uracil-targeting proteins in Archaea showed that this tendency is not restricted to M. acetivorans, but is prevalent and diverse in most Archaea. This work further underscores the importance of uracil-removal systems to maintain genome integrity in Archaea, including ‘UDG lacking’ organisms.
Highlights
Base deamination is one of the most commonly occurring forms of damage to DNA1
Our in vitro data indicate that the mesophilic euryarchaeon M. acetivorans appears to have developed a different strategy to counteract uracil damage to DNA with at least three distinct enzymes: endonuclease Q (EndoQ), exonuclease III (ExoIII), and family-4 uracil-DNA glycosylase (UDG)
Based on data from a previous study demonstrating a correlation between bacterial genes and those present in the M. acetivorans genome[53], we found that none of the proteins examined in this work (MacExoIII, MacEndoQ, MacUDG, and MacUDG-like) was acquired by horizontal gene transfer from bacteria, indicating that these proteins evolved in the archaeal domain and/or ancestor
Summary
Base deamination is one of the most commonly occurring forms of damage to DNA1. The hydrolytic deamination of cytosine, adenine, and guanine generates uracil, hypoxanthine, and xanthine, respectively, which are generated spontaneously under physiological conditions[1]. UDG family 2 is widespread in Bacteria and Eukarya, but shows more limited conservation compared to family 1 and is highly specific for mismatches such as U:G and T:G (thymine can be formed by the deamination of 5-methylcytosine)[19,20]. UDG family 4, known as thermostable UDG, is widespread in the archaeal domain and is found in some thermophilic bacteria These enzymes can remove uracil from both ssDNA and dsDNA20,21. As the major human AP endonuclease, ExoIII ( called APE1) has been described to exhibit cleavage activity toward uracil, but is substantially less effective than Mth21229 This type of AP endonuclease-initiated repair is commonly known as nucleotide incision repair[30,31]
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