Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease.
Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.
de la Campa
A G
AG
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Springhorn
S S
SS
Kale
P
P
Lacks
S A
SA
D001483:Base Sequence; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
D004262:DNA Restriction Enzymes; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific