I.R. Lehman

Branched DNA molecules: Intermediates in T4 recombination☆

Abstract Multiple infection of Escherichia coli with T4 phages defective in both DNA polymerase and ligase leads to the accumulation of DNA structures that are intermediates in recombination. When examined with the electron microscope, up to 25% of these phage DNA molecules contained one or more double-stranded branches of varying lengths and arrangements. Infection at a multiplicity of one failed to give rise to branched molecules. Differences in the frequency of the branched molecules were observed when the infecting phage carried genetic defects in addition to those in polymerase and ligase. DNA of hybrid density extracted from bacteria mixedly-infected with 13 C 15 N 32 P-labeled and 3 H-labeled T4 polymerase-ligase mutants was enriched in branched molecules, indicating that the branches were generated in the course of DNA exchange. These findings have suggested a molecular mechanism for recombination based on a process termed “branch migration”, in which there is a concerted dissociation of two sets of homologous parental DNA double helices and reassociation of two sets of hybrid recombinant complements, with a resulting displacement of the branch point sequentially along the interacting DNA molecules. Strand exchange is thus effected without the necessity of exposing long stretches of single-stranded DNA.

An in vitro transversion by a mutationally altered T4-induced DNA polymerase☆

Abstract In a reaction in which poly dC serves as template, highly purified T4 DNA polymerase has been shown to incorporate dTTP into poly dG, at a level ( T G ) of 10−5 to 10−6. The polymerase from a temperature-sensitive mutagenic strain of T4 with a mutation in the structural gene for DNA polymerase, substitutes T for G at a frequency about fourfold higher than the wild-type enzyme. The error frequency for both enzymes depends on dTTP and dGTP concentrations and can be increased about 5- to 20-fold by the substitution of manganese for magnesium in the reaction.

Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase☆☆☆

TAUts7 an Escherichia coli 15 strain with a thermolabile DNA ligase, has previously been shown to be a temperature-sensitive conditional lethal mutant that is sensitive to methyl methane sulfonate and to ultraviolet irradiation; it also accumulates 10 S DNA fragments to an abnormal extent. When the ligase mutation is transferred to a wild-type E. coli K12 strain, the strain becomes temperature sensitive for growth and displays the same characteristics as TAUts7. These findings show that a functional DNA ligase is essential for normal DNA replication and repair in E. coli.

Excision Repair of Uracil Incorporated in DNA as a Result of a Defect in dUTPase

Mutants of Escherichia coli that are severely defective in the enzyme dUTPase (dut) accumulate short (4 to 5 S) Okazaki fragments following brief pulses with [3H]thymidine. The transient appearance of DNA fragments in these mutants is plausibly explained by the misincorporation of uracil in DNA as a result of an increase in available dUTP, followed by its rapid excision and repair. The evidence in support of this interpretation is the following: (1) accumulation of short DNA fragments can be partially suppressed by a mutation in dCTP deaminase, presumably by decreasing the intracellular level of dUTP relative to dTTP; (2) accumulation of the short DNA fragments can be almost completely suppressed by a mutation in uracil N-glycosidase, probably by preventing the introduction of nicks at the sites of uracil incorporation; (3) introduction of DNA polymerase I or DNA ligase mutations into dUTPase-defective strains results in the persistence of the 4 to 5 S fragments and rapid cessation of DNA synthesis. Uracil N-glycosidase, DNA polymerase I and DNA ligase must therefore be involved in the excision repair of uracil-containing DNA.

The use of ethidium bromide-circular DNA complexes for the fluorometric analysis of breakage and joining of DNA☆

A new fluorometric method suitable for the analysis of cleavage and synthesis of phosphodiester bonds in closed circular duplex DNAs is described. The method depends upon the difference in the quantity of the intercalating dye, ethidium bromide, bound to fully closed circular DNA as compared with the same DNA which contains one or more single-strand breaks. The fluorometric method is extremely sensitive and can, for example, detect concentrations of pancreatic DNase as low as 2.5 × 10−5 μg/ml. Under properly standardized conditions it is also highly precise. Comparison of the kinetics of phosphodiester bond cleavage catalyzed by pancreatic DNase and Escherichia coli endonuclease I suggests that endonuclease I, unlike the pancreatic enzyme, may possess an exonucleolytic component. Similarly, an examination of the kinetics of phosphodiester bond synthesis by the E. coli joining enzyme indicates that the two breaks in cyclic DNA (“Hershey circles”) may be sealed at different rates. The fluorometric method can also be used to determine the molecular weight of any closed circular duplex DNA. Theoretical models are derived for the kinetics of breakage, joining as well as simultaneous breakage and joining of phosphodiester bonds in closed circular duplex DNA.

Enzymic joining of polynucleotides: III. The polydeoxyadenylate · polydeoxythymidylate homopolymer pair

Abstract The joining of oligomers (50 to 300 residues) of deoxythymidylate or deoxyadenylate by the polynuoleotide-joining enzyme from Escherichia coli occurs only when they are hydrogen-bonded to the complementary chain. The corresponding polyribonucleotides can neither be joined nor promote joining of the complementary polydeoxyribonucleotides. The rate and extent of joining of dT or dA oligomers are determined by the length of the complementary chain and the relative molar concentrations of deoxyadenylate and deoxythymidylate residues. Translational movement of dT oligomers relative to a dA chain occurs during the course of the reaction. There appears to be no significant exchange of oligomers between chains.

Enzymic joining of polynucleotides: VIII. Structure of hybrids of parental T4 DNA molecules

Abstract The structures of the “joint” (hydrogen-bonded) and “recombinant” (covalently-linked) hybrids of parental phage DNAs formed after infection of Escherichia coli strain BB with T4 amber mutants defective in the DNA polymerase or in the DNA polymerase and polynucleotide ligase genes were examined. The mixture of joint and recombinant DNA isolated after infection with DNA polymerase mutants consisted of molecules whose approximate size was one-fourth that of the parental T4 DNA and contained an average of two single-strand interruptions per molecule. The joint molecules isolated after infection with mutants defective in both the DNA polymerase and ligase were also one-fourth the size of the parental DNA; however, these contained an average of six single-strand interruptions per molecule. In both instances the interruptions were found to be gaps with mean lengths of 300 to 400 nucleotide residues.

Enzymic joining of polynucleotides: VII. Role of the T4-induced ligase in the formation of recombinant molecules

Abstract After mixed infection of Escherichia coli strain BB with 32P-labeled- and bromouracil density-labeled T4 am EB6 (a T4 mutant defective in the DNA polymerase gene), two kinds of hybrid molecules were isolated. One type contained the 32P- and bromouracil-labeled components linked only by hydrogen bonds (joint molecules); the second type contained the labeled components in covalent linkage (recombinant molecules). Infection with 32P-labeled- and bromouracillabeled T4 am EB6-605 (a T4 mutant defective in both the DNA polymerase and polynucleotide ligase genes) led to the formation of only joint DNA molecules. These results indicate that a functional T4-induced ligase is required for the formation of recombinant DNA molecules in vivo. Conversion of joint to recombinant molecules was achieved in vitro and required ligase, DNA polymerase and the four deoxynucleoside triphosphates. This finding suggests that joint molecules are double-stranded structures in which single-strand gaps separate the polynucleotide segments derived from the parental DNA molecules.

Enzymic joining of polynucleotides: IV. Formation of a circular deoxyadenylate-deoxythymidylate copolymer

The polynucleotide-joining enzyme from Escherichia coli catalyzes an intra-molecular joining reaction with linear deoxyadenylate-deoxythymidylate oligomers leading to the formation of single-stranded circular molecules. The rate of circle formation from oligomers in the range of 34 to 60 nucleotide residues increases with increasing chain length and temperature. The smallest circle thus far synthesized is composed of 34 to 36 nucleotides.

DNA Synthesis in Strains of Escherichia coli K12 with Temperature-sensitive DNA Ligase and DNA Polymerase I

Abstract The net DNA synthesis that persists at the restrictive temperature in the conditional lethal DNA ligase mutant Escherichia coli lig ts7 is semiconservative, suggesting that although the rate of joining of 10 S “Okazaki fragments” in the mutant is greatly reduced, it is nevertheless sufficient to permit continued progression of the replication fork and the initiation of new rounds of replication. DNA synthesis in E. coli lig ts7 of a phage (P2) that replicates its chromosomes unidirectionally is discontinuous on both strands. A double mutant of E. coli K12, ( lig ts7 pol A12), has been constructed which bears a temperature-sensitive mutation ( pol A12) in the structural gene for DNA polymerase I in addition to the lig ts7 mutation. Joining of the Okazaki fragments in the double mutant occurs at a slower rate than in either the lig ts7 or pol A12 parents. In contrast to the behavior of the single mutants, DNA synthesis in the double mutant stops abruptly upon shift from 25 °C to 42 °C.