Figure 4. Cas4 family proteins are 5′-3′ exonucleases.

A. Sso0001 (0.6 µM) cleaved the 5′ end labelled DNA 29mer (1 µM) in the presence of 10 mM Mg²⁺ into products with a size around 4 nt without any observable intermediates. In contrast, the 3′ end labelled DNA 29mer (1 µM) was cleaved to generate a ladder of progressively smaller products over the course of the reaction time in the presence of 10 mM Mg²⁺. Time points for both substrates were 0, 0.5, 1, 2, 3, 5, 10 and 15 min. The reaction temperature was 55°C. B. Sso0001 (0.3 µM) was incubated with 3′ end labelled DNA 50mer (500 nM) in reaction buffer at 55°C and products were analysed at 1, 2, 3, 5 and 10 min. In a parallel reaction (lanes 6–10), a 20 fold excess (10 µM) of unlabeled oligonucleotide of the same sequence was added to the reaction at the 1 min time point. A marked decrease in cleavage of the labelled oligonucleotide was observed, suggesting that Sso0001 is not highly processive, but can dissociate from substrates following each round of catalysis.