Catalytic GIY-YIG domain of type II restriction endonucleases R.Eco29kI, R.Cfr42I, and similar proteins
This family corresponds to the catalytic GIY-YIG domain of a group of GGCGCC-specific type II restriction endonucleases R.Eco29kI, R.Cfr42I, and similar proteins. R.Eco29kI is encoded on plasmid pECO29 in the E. coli strain 29K. This enzyme recognizes the palindromic 5'-CCGC/GG-3' target and cuts between Cyt4 and Gua5 on each strand of the restriction site to generate 3'-staggered ends. R.Eco29kI forms a domain-swapped homodimeric catalytically active complex during DNA binding and cleavage. Each subunit contains one GIY-YIG catalytic motif. Restriction endonucleases R.Cfr42I is an isoschizomer of R.Eco29kI. Unlike R.Eco29kI, R.Cfr42I is functional as a homotetramer, binding and cleaving two cognate DNA molecules in a cooperative manner. Members in this family are single-domain proteins sharing sequence similarities with the catalytic domain of GIY-YIG endonucleases, such as homing endonuclease I-TevI. However, they utilize loop insertions and terminal extensions instead of the separate DNA-binding domain to interact with the target site 5'-CCGC/GG-3'. A divalent metal-ion cofactor is required for their catalysis, but not for substrate binding. This family also includes a hypothetical protein from Deinococcus radiodurans that corresponds to MraI, a type II restriction enzyme similar to GIY-YIG family of homing endonucleases. MraI is shown to be an isoschizomer of Eco29kI, Cfr42I recognizing the palindromic nucleotide sequence 5'-CCGC reduced GG-3'. The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.
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