Sanford A. Lacks

Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation

Abstract Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5′-G-A-T-C-3′. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn 1 and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

Nuclease detection in SDS-polyacrylamide gel electrophoresis☆

Abstract Nucleases can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels containing DNA or RNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzymatic activity. Apparently, the acrylamide gel-nucleic acid combination provides an ideal environment for the removal of SDS and protein renaturation. The enzymes appear as sharp dark bands on a fluorescent background of ethidium bromide bound to unhydrolyzed nucleic acid. Several purified DNases and RNases, including basic proteins, have been detected. The major endonuclease of Diplococcus pneumoniae , a hydrophobic membrane protein, and the major exonuclease have been identified in gels upon electrophoresis of crude extracts of pneumococcal cells. Crude extracts prepared by lysis with SDS of Escherichia coli, Haemophilus influenzae, Bacillus subtilis , and HeLa cells have also revealed multiple bands of DNases and RNases. This demonstrates the wide applicability of the technique.

Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.

Molecular fate of DNA in genetic transformation of Pneumococcus.

Extracts of pneumococcal cells which had incorporated [ 32 P]DNA were fractionated in order to determine the fate of the incorporated radioactivity. Immediately after entry, about half of the DNA was found to be converted to a single-stranded form; the other half was degraded to dialysable oligonucleotides and inorganic phosphate. Very little native [ 32 P]DNA was present. On incubation of the cells for various lengths of time after introduction of the DNA, radioactivity was rapidly incorporated into the native DNA fraction. The sources of this radioactivity were (1) the fragments initially present which apparently were incorporated by means of normal synthetic processes, and (2) the single-stranded DNA some of which may have been transferred intact. This latter transfer appears to be the route by which genetic information is transferred. The increase of radioactivity in native DNA and the depletion of the single-stranded DNA corresponded in time with recovery of a genetic factor introduced by the donor DNA. The extent to which this genetic factor was recovered, determined both by direct measurement of its transforming activity and by measurement of the frequency of transformed cells, corresponded to a physical integration of about one-quarter of the donor DNA taken up by the cells.

Genetic and structural characterization of endA: A membrane-bound nuclease required for transformation of Streptococcus pneumoniae*

The endA gene encoding the membrane nuclease of Streptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying the endA+ gene complemented cells with endA- in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end. In vitro synthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity of endA and the effect of various insertions and deletions, it appears that endA is the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.

Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae

The susceptibility to repair of particular base mismatches by the hex system of Streptococcus pneumoniae was examined by comparison of the nucleotide sequence of the wild-type and eight mutant alleles of the malM gene. A detailed restriction map was constructed for pLS70, and the nucleotide sequence was determined for its 3475 bp chromosomal insert, which contains the entire malM gene (encoding amylomaltase), portions of malX and malP (encoding a membrane protein and a phosphorylase, respectively) and a control region. Transition mismatches were highly susceptible to repair; transversion mismatches, much less so. A mismatch caused by a single-nucleotide deletion was reparable, but mismatches with longer deletions were not. The hex system also reduced spontaneous reversion of mutations corresponding to transitions. It is suggested that recognition of donor or nascent DNA strands by the hex system depends on single-strand breaks in the target strand, and that the role of DNA methylation in mismatch repair of Escherichia coli can be accommodated to this model.

Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of Streptococcus pneumoniae bound to S-adenosylmethionine

BACKGROUND . Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. RESULTS . The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. CONCLUSIONS . DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.

Complementary action of restriction enzymes endo R · DpnI and endo R · DpnII on bacteriophage f1 DNA

Abstract Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks & Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam+ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam− cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam +) and one unmethylated strand (dam−) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.

Fate of donor DNA in pneumococcal transformation

Abstract The molecular fate of homologous (pneumococcal) and heterologous ( Escherichia coli ) [ 32 P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 32 P-containing products at the earliest time of observation was the same. About 50% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32 P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. coli as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32 P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and l -α-glycerophosphate and smaller amounts of the four 5′-deoxynucleotides. A plausible origin of labeled inorganic phosphate is the dephosphorylation of nucleotides. The labeled glycerophosphate, in turn, may be derived from inorganic phosphate, since artificially introduced [ 32 P]phosphate rapidly equilibrated with a large pool of intracellular glycerophosphate. Thus, the initial products of entry are envisioned to be equal amounts of single strands and 5′-deoxynucleotides. This would be compatible with their origin in the degradation by a pneumococcal exonuclease of one strand of an incoming DNA duplex. On subsequent incubation, single strands of heterologous origin were slowly degraded; their breakdown products were used for de novo DNA synthesis. The disappearance of homologous single strands was much faster and corresponded to an increase of 32 P in native DNA as well as to the recovery from eclipse of donor marker-transforming activity. It is concluded that single strands are precursors of genetically integrated DNA and that recombination involves the interaction of homologous single strands with host DNA. The ratio of genetically integrated DNA to total DNA incorporated suggests that only half, or somewhat more, of the single-stranded DNA was inserted as intact sequences. Inhibition of DNA synthesis by aminopterin gave results which were compatible with these conclusions.

Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis.

The extent of renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate depended on the source of the detergent. Analysis of commercial preparations of sodium dodecyl sulfate revealed appreciable amounts of tetradecyl and hexadecyl sulfates in some preparations. Inhibition of renaturation was correlated with the amount of hexadecyl sulfate and, to a much lesser extent, of tetradecyl sulfate present. The higher alkyl sulfates appeared to bind more tenaciously to proteins in the gel. More extensive washing was required to remove them than to remove dodecyl sulfate, and they were inhibitory to enzyme activity at lower detergent concentrations. A system is described for gas chromatographic analysis of alkyl sulfates containing chains of 10 to 16 carbon atoms in length.