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

Konrad, E. Bruce; Modrich, Paul; Lehman, I. R.

doi:10.1016/0022-2836(73)90220-9

Cited by 128 publications

(63 citation statements)

References 15 publications

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“…After centrifugation for 15 min at 15,000 X g, the supernatant solution was removed and stored at -20°. Alkaline sucrose gradients (5-20%) were run as described by Konrad et al (12), except that an SW 41 rotor was used and gradients were formed over a l-ml shelf of alkaline 80% sucrose. A portion (25O-,M) of DNA extract was layered onto the gradient and covered with mineral oil.…”

Section: Methodsmentioning

confidence: 99%

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

Konrad

Lehman

1974

Proc. Natl. Acad. Sci. U.S.A.

141

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A mutant strain of E. coli, initially identified by an abnormally high frequency of recombination, has been found to be defective in the 5' -. 3' exonuclease associated with DNA polymerase I, but not in the polymerase activity. This defect is tolerated at 300, but is lethal at 43°. Like other polymerase I mutants, the strain is unusually sensitive to methyl methanesulfonate and to ultraviolet irradiation; it is also unable to support the growth of phage X defective in general recombination, and shows a reduced rate of joining of 10S "Okazaki fragments." These results demonstrate that a functional DNA polymerase I is essential for normal growth and viability in E. coli K12.Escherichia coli K12 strains defective in the structural gene for DNA polymerase I (polA) have been extensively described (1-5). These mutants are abnormally sensitive to ultraviolet irradiation and to the alkylating agent methyl methanesulfonate, and show retarded joining of the 10S "Okazaki fragments" (6)(7)(8). A recent analysis of partially purified fractions derived from..several of the polA mutants has demonstrated that although they contain much reduced levels of DNA polymerase I activity the 5' --3' exonuclease associated with the polymerase I is present in nearly normal amounts (9).We have found that polA mutants are also characterized by an increased frequency of recombination at least under some conditions (7). By screening mutagenized cells for this phenotype, we have isolated a novel polA mutant (polAexl) that we describe in this paper. This mutant has a normal level of polymerase I activity but a greatly reduced level of 5' -_ 3' exonuclease. Unlike other polA mutants, it is conditionally lethal. MATERIALS AND METHODSBacterial and Phage Strains. All bacterial strains are derived from E. coli K12. Their origin and genotypes are given in Table 1. Phage X defective in general recombination (A red 3) was provided by D. Freifelder.Microbiological Procedures. Pivir phage stocks were prepared and used for transduction, and Hfr crosses were performed as described by Miller (10). Sensitivity to ultraviolet irradiation was determined as follows. Cells grown at 30°in H broth (11) to A595 = 0.5 were chilled, harvested, and resuspended in an equal volume of chilled M63 medium (10); 10 ml of a 5-fold dilution of this suspension in a glass petri plate was irradiated (25 ergs per mm2 per sec) with a General Electric germicidal lamp; and samples were removed at various times, diluted in chilled H broth, and plated on yeast extract-tryptone plates (10) that were incubated at 300 in the dark. Sensitivity to methyl methanesulfonate was determined on fresh yeast extract-tryptone plates supplemented with 0.04% methyl methanesulfonate. Cultures were treated with ethyl methanesulfonate as described by Miller (10). Lactosetetrazolium indicator plates were prepared according to Miller (10). Ability to form plaques of phage A was determined on tryptone plates as described by Miller (10). Unless otherwise noted, E. coli polAexl strains were grown at ...

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Section: Methodsmentioning

confidence: 99%

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

Konrad

Lehman

1974

Proc. Natl. Acad. Sci. U.S.A.

141

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“…A conditional mutant of Escherichia coli LigA results in growth arrest at the restrictive temperature; thus LigA is essential in E. coli (8,9). LigA is also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (10 -12).…”

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Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Gong

Martins

Bongiorno

et al. 2004

Journal of Biological Chemistry

129

142

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Mycobacterium tuberculosis encodes an NAD ؉ -dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD ؉ and ATP, respectively, whereas LigC and LigD, which have ATPspecific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA- DNA ligases are grouped into two families, ATP-dependent ligases and NAD ϩ -dependent ligases, according to their nucleotide substrate requirement (1, 2). The ligase reaction entails three nucleotidyl transfer steps (1). In the first step, attack by ligase on the ␣ phosphorus of ATP or NAD ϩ results in release of PP i or NMN and formation of a covalent ligase-adenylate intermediate. In the second step, the AMP is transferred to the 5Ј end of the 5Ј phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by a DNA 3Ј-OH on DNA-adenylate to join the two polynucleotides and liberate AMP. ATP-dependent DNA ligases are found in all three domains of life (Bacteria, Archaea, and Eukarya), whereas the NAD ϩ -dependent enzymes are present only in bacteria and entomopoxviruses (1-7).All known bacteria encode a highly conserved NAD ϩ -dependent DNA ligase (LigA). A conditional mutant of Escherichia coli LigA results in growth arrest at the restrictive temperature; thus LigA is essential in E. coli (8,9). LigA is also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (10 -12). Some bacteria, including E. coli, S. typhimurium, Shigella flexneri, Yersinia pestis, and Pseudomonas putida, have a second NAD ϩ -dependent ligase (13), the function of which is not known.The presumption that bacteria encode only NAD ϩ -dependent DNA ligases was overturned by the demonstration in 1997 of an ATP-dependent ligase in the respiratory pathogen Haemophilus influenzae (5). The 268-amino acid (aa) 1 H. influenzae ligase consists of a minimized catalytic domain. ATP-dependent ligase homologues coexist with NAD ϩ -dependent enzymes in several other bacterial species, including major human pathogens such as Neisseria meningitidis, Y. pestis, Vibrio cholerae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (3,14,15).Remarkably, M. tuberculosis encodes three distinct ATP-dependent ligase homologues (LigB, LigC, and LigD) plus an NAD ϩ -dependent ligase (LigA) (Fig. 1) (16). To begin to understand the rationale for this plethora of ligases in a single bacterium, we have produced and characterized recombinant versions of the mycobacterial DNA ligase enzymes and probed genetically the essentiality or dispensability of the ATP-dependent ligases for growth of M. tuberculosis. EXPERIMENTAL ...

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“…The NAD ϩ ligases lack a recognizable counterpart of motif VI within their OB-fold domains, which is sensible given that the NAD ϩ substrate does not contain a ␥ phosphate. Early genetic studies showed that the ligA gene is essential for growth of E. coli (17,18). Genes encoding NAD ϩ -dependent LigA are also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (19 -21).…”

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Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

Sriskanda

Shuman

2002

Journal of Biological Chemistry

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NAD؉ -dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD ؉ ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD ؉ and then to isolate small molecules that recognize these components and thereby block the binding of NAD ؉ to the ligase. The limitation to this strategy is that the structural determinants of NAD ؉ specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD ؉ requires N-terminal domain Ia, which is unique to, and conserved among, NAD ؉ ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3-OH/5-PO 4 nicks and the reaction with NAD؉ to form ligaseadenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5-PO 4 nick and adenylyl transfer from NAD ؉ without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD ؉ . Structure-activity relationships were clarified by conservative substitutions. DNA ligases are grouped into two families, ATP-dependent ligases and NADϩ -dependent ligases, according to the cofactor required for ligase-adenylate formation (1). Both types of DNA ligases catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three sequential nucleotidyl transfer reactions. In the first step, attack on the ␣ phosphorus of ATP or NAD ϩ by ligase results in release of pyrophosphate or nicotinamide mononucleotide (NMN) 1 and formation of a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide bond to lysine. In the second step, the AMP is transferred to the 5Ј end of the 5Ј-phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by the 3Ј-OH of the nick on DNA-adenylate to join the two polynucleotides and release AMP.

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

Cited by 128 publications

References 15 publications

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

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