The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites
| Autor: | Mark D. Szczelkun, Fiona M. Diffin, Kara van Aelst, Eva Šišáková |
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| Rok vydání: | 2012 |
| Předmět: |
Protein domain
Cleavage (embryo) Potassium Chloride 03 medical and health sciences chemistry.chemical_compound Endonuclease Plasmid Recognition sequence Genetics DNA Cleavage 030304 developmental biology 0303 health sciences biology 030306 microbiology Nucleic Acid Enzymes Lactococcus lactis Helicase DNA DNA Restriction Enzymes DNA Methylation biology.organism_classification Protein Structure Tertiary chemistry Biochemistry biology.protein |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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