Figure EV2. Activity dependence on divalent metal ions and sequence features.

1. Integration reactions were performed in the presence of different divalent metal co‐factor using Cas1:Cas2‐DnaQ complex and 23 bp DNA duplex with 5 nt 3′‐overhangs as protospacer. Mg²⁺, Mn²⁺, and Ca²⁺ were determined to be the best cofactors for integration reaction. Mn²⁺ activates DnaQ domain so strongly that integration becomes undetectable, due to degradation of spacers. Reactions were incubated for 30 min at 42°C.
2. Exonuclease reactions were performed in the presence of different divalent metal co‐factor using Cas1:Cas2‐DnaQ complex and 72 nt single‐stranded DNA oligonucleotide. DNA degradation was most efficient with Mg²⁺ and Mn²⁺ ions.
3. Exonuclease rates of Cas1:Cas2‐DnaQ complex were probed using polyA, polyT, and polyC single‐stranded DNA oligonucleotides.
4. Exonuclease rates of Cas1:Cas2‐DnaQ complex were probed using polyA oligonucleotide (2′dT), which was blocked at its 3′‐end with 2′, 3′‐dideoxyadenosine (2′d3′dA) or 3′‐deoxyadenosine (3′dA) using terminal deoxynucleotidyl transferase.