Phylogenetic analysis and in vitro bifunctional nuclease assay of arabidopsis BBD1 and BBD2

Nucleases are a very diverse group of enzymes that play important roles in many crucial physiological processes in plants. We previously reported that the highly conserved region (HCR), domain of unknown function 151 (DUF151) and UV responsive (UVR) domain-containing OmBBD is a novel nuclease that does not share homology with other well-studied plant nucleases. Here, we report that DUF151 domain-containing proteins are present in bacteria, archaea and only Viridiplantae kingdom of eukarya, but not in any other eukaryotes. Two Arabidopsis homologs of OmBBD, AtBBD1 and AtBBD2, shared 43.69% and 44.38% sequence identity and contained all three distinct domains of OmBBD. We confirmed that the recombinant MBP-AtBBD1 and MBP-AtBBD2 exhibited non-substrate-specific DNase and RNase activity, like OmBBD. We also found that a metal cofactor is not necessarily required for DNase activity of AtBBD1 and AtBBD2, but their activities were much enhanced in the presence of Mg²⁺ or Mn²⁺. Using a yeast two-hybrid assay, we found that AtBBD1 and AtBBD2 each form a homodimer but not a heterodimer and that the HCR domain is possibly crucial for dimerization.