The activity, divergence, and evolutionary degradation of modern-day homing endonucleases and behavior of their reconstructed ancestors.

Homing endonucleases (HEs) are selfish genetic elements that drive the mobilization of their own coding sequences, often in concert with surrounding introns. Homing endonuclease genes (HEGs) usually display life cycles in which they accumulate inactivating mutations after invading a host genomic target site, leading to eventual removal from the genome. We identified several hundred novel HEGs and determined the distribution of their proteins' behaviors and activities. Approximately 10% are expressed as well-folded functional proteins that cleave predictable DNA target sites. Another ∼20% display significant expression but little to no cleavage activity; the remainder display severely reduced expression. Despite the presence of debilitating mutations throughout most HEGs, ancestral reconstructions yielded endonucleases with improved expression and stability. One such reconstructed model, at a hypothetical node preceding highly diverged HEs that cleave unique target sites, binds (but does not cleave) their individual targets. It instead cleaves a DNA sequence that resembles a hybrid of those modern-day DNA targets, while displaying a similar specificity profile to previously characterized wild-type HEs. Its DNA-bound crystal structure adds detail to our understanding of how HE DNA contacting surfaces and residues shift and rearrange during evolution, ultimately leading to their action at new target sites.