title: The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites.
abstract: The Type ISP Restriction-Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5'-TnAGCC-3', where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K(+) ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation-collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K(+) ions produces a far more distributive cleavage profile.
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Eva
authors.initials: E
authors.lastname: Šišáková
authors.forename: Kara
authors.initials: K
authors.lastname: van Aelst
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Diffin
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D011189:Potassium Chloride / Q000737:chemistry; D017434:Protein Structure, Tertiary
chemical_list: D011189:Potassium Chloride; D004247:DNA; D004262:DNA Restriction Enzymes