DNA Nucleotide Sequence Restricted by the RI Endonuclease

Abstract

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage X has been determined. The 5'-terminal nucleotide labeled with 32p and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in X DNA was deduced from these data and from the 3'-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virusThe RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5'-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry.The restriction and modification enzymes of several host specificities (1-7), including the endonuclease and methylase of the host-specificity (RI) controlled by the fi+ R-factor (Yoshimori, Roulland-Dussoix, Aldridge & Boyer; Yoshimori, Roulland-Dussoix, Goodman & Boyer, to be published) have been characterized. The restriction endonuclease of a given host specificity interacts with a specific sequence of DNA base-pairs and produces a double-strand break in the molecule (1-6), while the modification methylase methylates a base in each strand of this sequence (refs. 3 and 7; Yoshimori, Roulland-Dussoix, Goodman & Boyer, to be published). Methylation of a base in either strand is sufficient to prevent the endonuclease from attacking the sequence (3). Therefore, in molecular terms, a given DNA restriction and modification host-specificity can be defined by the sequence of base pairs recognized by the restriction endonuclease and modification methylase. In this paper, we present an analysis of the sequence of base pairs adjacent to the phosphodiester bond cleavages made in bacteriophage X DNA by the RI restriction endonuclease. MATERIALS AND METHODSEnzyme. The RI restriction endonuclease, purified as will be described by Yoshimori, Roulland-Dussiox, Aldridge, and Boyer (to be published), was free of detectable nonspecific exo-or endonuclease activities and migrated as one band in native or Na dodecyl S04-polyacrylamide gels. Purified RNAdirected DNA polymerase of Rous Sarcoma virus, free of detectable exo-or endonucleolytic activities (8,9), was a generous gift from Dr. A. J. Faras. Polynucleotide kinase (10) was purified as described elsewhere. Escherichia coli alkaline phosphatase rechromatographed as described elsewhere (11), pancreatic DNase, micrococcal nuclease, venom phosphodiesterase, and spleen phosphodiesterase were obtained from Worthington Biochemical Co.Phage X DNA. A X (CI857SUSS7) lysogen of E. coli 1100 was used as a source of X phage. Tryptone broth cultures of lysogens (2 X 108 cell per ml) were heated to 420 for 0.5 hr (isotope was added here for labeled DNA) and incubated at 370 for 3 hr. The cells were collected by low-speed centrifugation and resuspended in 5-10 ml of 50 mM Tris-HCl (pH 7.5)-10 mM MgCl2. 0.5 ml of CHC13 was added and ...

The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease

“…This result was first reported for bacteriophage A DNA by Thomas and Davis [5] and investigated in more detail later also by Halford et al [6] and Berkner and Folk [7], who showed that the five EcoRI sites in bacteriophage A DNA are cleaved by the EcoRI endonuclease with different rates. Similar results were obtained later by Goldstein et al [8] with bacteriophage T4 DNA and by Forsblom et al [9] with adenovirus DNA. Rubin and Modrich [lo] have pointed out that the differences in the rate of doublestrand cleavage may be related to the differences observed in the rate of accumulation of the intermediate containing a single-strand break.…”

Size and Base Composition of RNA in Supercoiled Plasmid DNA

“…The results of an experiment involving a CM-ColE1 DNA preparation in which 32% of the molecules were sensitive to nicking by alkali are shown in Table 2. The total radioactivity released represented 0.25% of the RNA-containing CM- ColE1 DNA, but that in the four nucleotide spots comprised 0.21% of these molecules, equivalent to an average of 26 ribonucleotides per RNA-containing plasmid molecule. The proportions of the individual ribonucleotides are shown in Table 2.…”

“…Confirmation of the average ribonucleotide composition determined by electrophoretic methods was achieved by two procedures previously used to analyze the single-strand termini generated by the E. coli RI and RII restriction endonucleases (24,26). The ribonucleotides that confer RNase sensitivity on supercoiled CM-ColE1 molecules were first removed by digestion with RNase H plus RNase A.…”

Repetitive primate DNA containing the recognition sequences for two restriction endonucleases which generate cohesive ends

“…and Hind Ill endonucleases. Such fragments bear cohesive ends [8][9][10][11] and can serve as useful 'linkers' in molecular engineering experiments where DNA sequences of different origins bearing either the Eco R I or the Hind III termini are to be joined with each other.…”