Domain organization and functional analysis of type IIS restriction endonuclease Eco31I.
Type IIS restriction endonuclease Eco31I harbors a single HNH active site and cleaves both DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). A two-domain organization of Eco31I was determined by limited proteolysis. Analysis of proteolytic fragments revealed that the N-terminal domain of Eco31I is responsible for the specific DNA binding, while the C-terminal domain contains the HNH nuclease-like active site. Gel-shift and gel-filtration experiments revealed that a monomer of the N-terminal domain of Eco31I is able to bind a single copy of cognate DNA. However, in contrast to other studied type IIS enzymes, the isolated catalytic domain of Eco31I was inactive. Steady-state and transient kinetic analysis of Eco31I reactions was inconsistent with dimerization of Eco31I on DNA. Thus, we propose that Eco31I interacts with individual copies of its recognition sequence in its monomeric form and presumably remains a monomer as it cleaves both strands of double-stranded DNA. The domain organization and reaction mechanism established for Eco31I should be common for a group of evolutionary related type IIS restriction endonucleases Alw26I, BsaI, BsmAI, BsmBI and Esp3I that recognize DNA sequences bearing the common pentanucleotide 5'-GTCTC.
Jakubauskas
Arturas
A
Institute of Biotechnology, Graiciuno g. 8, Vilnius LT02241, Lithuania. arturas.jakubauskas@santa.lt
Sasnauskas
Giedrius
G
Giedriene
Jolanta
J
Janulaitis
Arvydas
A
D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D004269:DNA, Bacterial; C047595:endodeoxyribonuclease Eco31I; D015252:Deoxyribonucleases, Type II Site-Specific