Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif, These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site, We showed that f our divergent endonucleases from the I-Crel subfamily cleave the same DNA s ubstrates, Mapping of the 66 amino acids that are conserved among the membe rs of this subfamily on the 3-dimensional structure of I-Crel bound:to its recognition sequence revealed that these residues participate in protein fo lding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-Crel amino acids interacting with DNA are conserved, sugg esting that I-Crel and its homologs use different subsets of residues to re cognize the same DNA sequence. Our sequence comparison of all 45 single-LAD LIDADG proteins identified so far suggests that these proteins share relate d structures and that there is a weak:pressure in each subfamily to maintai n identical protein-DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides ins ight into how these proteins evolve new DNA specificity.