Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease.
1988; Elsevier BV; Volume: 263; Issue: 29 Linguagem: Inglês
10.1016/s0021-9258(18)68093-7
ISSN1083-351X
AutoresAdela G. de la Campa, S S Springhorn, P.G. Kale, Sanford A. Lacks,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoInsertion 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 NH2terminal 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.Strains of Streptococcus pneumoniae can harbor one of two restriction systems, DpnI or DpnII (reviewed in Lacks et al., 1987).The two systems are complementary in specificity and mutually exclusive (Lacks and Greenberg, 1977; Muckerman et at., 1982).Both systems enable the cells to resist bacteriophage attack by reducing viral infectivity to (Muckerman et al., 1982).The genetic basis of the phenotypic variation in restriction systems in S. pneumoniae depends on a cassette mechanism, in which alternative gene clusters are positioned at a specific location in the chromosome (Lacks et al., 1986b).The DpnII cassette confers a typical restriction system in the sense that the restriction endonuclease recognizes and cleaves the DNA sequence 5'-GATC-3' when it is not methylated.However, the organization of genes in the DpnII cassette is not typical.The system consists of an operon composed of three genes (dpnM, dpnA, and dpnB) under
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