DNA cleavage by type III restriction-modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites

The type III restriction-modification enzyme EcoP151 requires the interacti on of two unmethylated, inversely oriented recognition sites 5'-CAGCAG in h ead to head configuration to allow an efficient DNA cleavage. It has been h ypothesized that two convergent DNA-translocating enzyme-substrate complexe s interact to form the active cleavage complex and that translocation is dr iven by ATP hydrolysis. Using a half-automated, fluorescence-based detectio n method, we investigated how the distance between two inversely oriented r ecognition sites affects DNA cleavage efficiency. We determined that EcoP15 1 cleaves DNA efficiently even for two adjacent head to head or tail to tai l oriented target sites. Hence, DNA translocation appears not to be require d for initiating DNA cleavage in these cases. Furthermore, we report here t hat EcoP151 is able to cleave single-site substrates. When we analyzed the interaction of EcoP151 with DNA substrates containing adjacent target sites in the presence of non-hydrolyzable ATP analogues, we found that cleavage depended on the hydrolysis of ATP. Moreover, we show that cleavage occurs a t only one of the two possible cleavage positions of an interacting pair of target sequences. When EcoP151 bound to a DNA substrate containing one rec ognition site in the absence of ATP, we observed a 36 nucleotide DNaseI-foo tprint that is asymmetric on both strands. All of our footprinting experime nts showed chat the enzyme did not cover the region around the cleavage sit e. Analyzing a DNA fragment with two head to head oriented recognition site s, EcoP151 protected 27-33 nucleotides around the recognition sequence, inc luding an additional region of 26 bp between both cleavage sites. For all D NA substrates examined, the presence of ATP caused altered footprinting pat terns. We assume that the altered patterns are most likely due to a conform ational change of the enzyme. Overall, our data further refine the tracking -collision model for type III restriction enzymes. (C) 2001 Academic Press.