Abstract
Targeted DNA degradation by the Type I CRISPR-Cas system is performed by Cas3, typically a two-component protein with a nuclease and a helicase domain. The nuclease belongs to the HD-domain metalloprotein superfamily, that harbors functionally diverse representatives including phosphodiesterases, phosphatases and oxygenases. The range of metal ions reported to support Cas3 catalytic activity is diverse, including Mg, Mn, and Ni, demonstrating variability in cofactor requirements. Type IE Cas3 nucleases are dinuclear, whereas for Type IA Cas3s (containing only the nuclease domain) assembly of a binuclear site has not been demonstrated. In the present study, we redefine the metal ions that confer hydrolytic activity and show that the di-ferrous cofactor in Thermobifida fusca Cas3 degrades ssDNA with the highest observed rates. We also demonstrate assembly of diiron cofactors in two Type IA Cas3s and show that these have phosphodiesterase activity. In addition, Cas3 efficiently hydrolyzes 2’3’-cAMP, in agreement with previous notions that it may act as a nucleotidase at the 2’3’-cyclic phosphate crRNA termini. 2’3’-cAMP hydrolysis is specific to Cas3, and not a general property of the HD-domain scaffold. ssDNA and 2’3’-cAMP cleavage exhibit the same metal-dependence, suggesting that cofactor chemical nature is not a substrate specificity element. HD-domain PDEs harbor a seventh conserved histidine ligand, for which the role remains unclear. We show that this histidine is not essential for activity, but is crucial for substrate (re)positioning, in particular for the larger ssDNA substrate compared to the smaller 2’3’-cAMP. We also show that Cas3’’ proteins can harbor a diiron site with a similar coordination environment as that of Tf Cas3 and suggest Cas3’’s utilizes the same seven residue HD-motif to assemble their active sites.