Figure 3. Cas4 family proteins are metal dependent nucleases.

A. Sso0001 (0.6 µM) cleaves the ssDNA oligonucleotide 15T (1 µM) in the presence of either Mg²⁺ or Mn²⁺ but the ssRNA oligonucleotide 20U (1 µM) only in the presence of Mn²⁺. Both oligonucleotides were 5′-end labelled with a 5′-fluorescein moiety. Reactions were carried out at 75°C for 10 min. B. The Sso0001 D99A variant does not cleave ssDNA oligonucleotide 15T (1 µM) in the presence of Mg²⁺ ions. Assays were carried out as in panel A with 0.6 or 1.2 µM of the relevant protein. Reactions were carried out at 75°C for 10 min. C. Circular phiX174 virion ssDNA (60 nM) was incubated with 1.2 µM wild-type or 3 µM D99A Sso0001 for 10, 20 and 40 min at 55°C in the presence of MgCl₂. No degradation of the DNA was observed, suggesting that Sso0001 requires a ssDNA end for activity. The control lane (C) was incubated in the same conditions in the absence of enzyme. dsDNA size markers (m) are indicated.