Communications between catalytic sites in the protein-DNA synapse by the SfiI endonuclease.
The SfiI endonuclease is a tetrameric protein with two DNA-binding clefts. It has to bind two copies of its recognition sequence, one at each cleft, before it cleaves DNA. While SfiI binds cooperatively to two cognate sites, it binds only one non-cognate DNA molecule at a time and the resultant complex is precluded from binding cognate DNA at the vacant cleft. To examine the communications between separate binding sites in a protein that synapses two segments of DNA, SfiI was tested with oligonucleotide duplexes containing its recognition sequence but with either R(p) or S(p) phosphorothioate linkages at the scissile bonds. Though SfiI has low activity on the R(p) and none against the S(p) diastereoisomer, it bound these duplexes in the same cooperative manner as oxyester duplexes, though with a reduced affinity for the S(p) derivative. It also formed complexes with one phosphorothioate-duplex and one oxyester-duplex but, when Mg(2+) was added to the hybrid complexes, the phosphorothioate moiety at one DNA-binding cleft prevented the enzyme from cleaving the oxyester duplex at the other cleft. SfiI is thus restrained from catalytic action until it recognises the correct nucleotide sequence at two DNA loci and the correct phosphodiester functions at both loci.
Williams
Shelley A
SA
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Halford
Stephen E
SE
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific