John B. Hays

T4 Endonuclease VII Cleaves Holliday Structures

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.

Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda.

The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene). A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed. Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages. The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability.

Expression of the phage λ recombination genes exo and bet under lacPO control on a multi-copy plasmid

Abstract The bacteriophage λ genes exo and bet, whose products (λ exonuclease and β protein, respectively; Red phenotype) mediate homologous recombination of λ phages, have been cloned under lacPO lacI q control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in λ exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of λ Red − phages in vivo was similarly inducible. Only one out of 25 bet Δ plasmids (constructed by a variety of in vitro techniques) expressed λ exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar ( bet 113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. When λ bet 3 phages (Exo − Bet − ) infected IPTG-induced recA 3 bacteria containing exo + bet + plasmids, recombination frequencies were no more than twice those typical for infection of plasmid-free recA 3 cells with exo + bet + phages, even in the case of IPTG induction sufficient to elevate the production of λ exonuclease about 100-fold. Even when plasmid induction was delayed till as late as 50 min after infection, recombination was significant. Preliminary experiments suggest that these plasmids encode a polypeptide with Gam activity that corresponds to the 98-amino acid “shorter” open reading frame assigned to gam by Sanger et al.

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA: I. Identification and mapping of arl mutations☆

Abstract Hyper-rec mutants of Escherichia coli were originally identified as lac -diploid strains whose colonies exhibited unusually high numbers of Lac + papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl . Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl + bacteria, but not in recA cells. Both the high frequency of Lac + papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT , do not confer a hyper-rec phenotype (by the lac -diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.

Irreversible inactivation of the membrane-bound enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus by triton X-100 and protection by substrates

Enzyme IIlac, the membrane-bound component of the lactose phosphotransferase system of Staphylococcus aureus, catalyzes the phosphorylation-transport reaction below: (see article). (The sugar can be lactose or one of its analogs.) The effects of the non-ionic detergents Triton X-100, Brij 35, and Tween 40 on the activity of Enzyme IIlac were studied. Especially striking effects were observed using Triton X-100, a detergent previously used to solubilize and isolate this enzyme. A systematic study of Triton effects over a range of concentrations and temperatures demonstrated three aspects of Triton-membrane interaction. At 0.1% Triton and 25 degrees C Enzyme IIlac is activated, but remains particulate. At 0.5% Triton and 0.5% Triton and 37 degrees C, it is rapidly and irreversibly inactivated. Sugar substrates and inhibitory sugar analogs protect Enzyme IIlac against inactivation; the effect is specific for beta-galactosides. The other substrate of Enzyme IIlac, phospho-Factor IIIlac, does not affect Triton inactivation, and the product analog galactose 6-phosphate slightly enhances the inactivation rate.

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA: V. Recombinogenic plasmids from arl mutants of Escherichia coli are unusually sensitive to nuclease S1 and partially deficient in cytosine methylation at C-C-(A/T)-G-G sequences☆

Two novel phenotypes previously associated with arl mutations of Escherichia coli, increased frequencies of genetic recombination and unusual sensitivity of DNA to the single-strand-specific nuclease S1, have been defined most completely by the properties of λ bacteriophages grown on arl bacteria (Arl− phages). We now find that plasmids maintained in arl mutants (Arl− plasmids) exhibit elevated recombination frequencies, unusual sensitivity to nuclease S1 (in a limited number of regions) and a new Arl phenotype, partially deficient methylation of the inner cytosine at C-C-(A/T)-G-G sequences. A variety of Arl− plasmids (all pBR322 derivatives) show elevated recombination (4 to 10-fold) by three different assays (frequencies of homomultimers and of heteromultimers, efficiency of intramolecular recombination). Plasmids from arl bacteria (after conversion to linear form) are nicked by nuclease S1 about 0.7 times per duplex; Arl+ plasmids are nuclease S1-resistant. Restriction endonuclease EcoRII (recognition sequence, C-C-(A/T)-G-G) cuts Arl− plasmid DNA more readily than Arl+ DNA, but Arl− plasmids are still more EcoRII-resistant than Dcm− plasmids (from E. coli dcm mutants, which lack the chromosomal cytosine methylase; recognition sequence, also C-C-(A/T)-G-G). By chromatographic analyses, Arl− plasmid DNA contains less 5-methylcytosine than Arl+ (0.07% versus 0.15%). although the 6-methyladenine content is the same (0.5mol%).

Novel mutants of Escherichia coli that produce recombinogenic lesions in DNA: II. Properties of recombinogenic λ phages grown on bacteria carrying arl mutations

Abstract Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl − phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl + bacteria. Arl − phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl − phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl − phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo ), and the same thermal denaturation profile as DNA from phages grown on arl + bacteria. Recombination of Arl − phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m 2 ) Arl − phages was more than twice the sum of the frequencies for unirradiated Arl − phages and irradiated control phages. Substantially increased recombination of Arl − phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III

SummaryThree aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function.Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutations

Generalized recombination in tandem duplications of bacteriophage lambda

SummaryRecombination between the tandem duplicated segments of λb221a106-15 yields unduplicated (“single-copy”) λb221 phage. The apparent frequency of intramolecular events among these recombinations was determined for both cellular (“Rec”) and bacteriophage (“Red”) generalized recombination systems. The progeny from single-cycle growth experiments with genetically marked duplication phages were treated with EDTA to inactivate all but the singlecopy phages produced by recombination. Analysis of the genotypes of the EDTA-resistant phages suggested that intramolecular events were about 1 to 5 times as frequent as intermolecular ones. While the results suggest that intramolecular events are not intrinsically forbidden, the quantitative values for the ratio depend on the assumption that intracellular phage chromosomes are completely mixed.

Studies on the mechanism of phosphorylation and transport of β-galactosides by the lactose phosphotransferase system of Staphylococcus aureus kinetic investigations using tosyl galactosides as reversible dead-end inhibitors

Tosyl galactosides, previously shown to be potent reversible dead-end inhibitors of the membrane-bound Enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus, were used for an investigation of the kinetic mechanism of the sugar phosphorylation/transport reaction catalyzed by this enzyme: phospho-Factor IIIlac&sugar Enzyme IIlac lead to Factor IIIlac&sugar phosphate. Inhibition of Enzyme IIlac was studied in three different systems. Washed membranes, and washed membranes in the presence of 0.1% Triton X-100 were used for phosphorylation experiments, and whole cells were used for transport studies. When washed membranes were used to supply Enzyme IIlac, inhibition of phosphorylation by tosyl galactoside was linear non-competitive against both the sugar and phospho-Factor IIIlac substrates, with an apparent Ki of about 0.5 mM. This Ki decreased with increasing Factor IIIlac concentration. In the presence of 0.1% Triton X-100, the phosphorylation reaction was stimulated; under these conditions the inhibition became strictly competitive against sugar, and completely uncompetitive against phospho-Factor IIIlac. Apparently washed membranes can catalyze phosphorylation both via a reaction sequence in which sugar binds first and via one in which phospho-Factor IIIlac binds first, but in the presence of 0.1% Triton the reaction does not occur by the former sequence. The inability of bound phospho-Factor IIIlac to hinder the binding of tosyl galactosides suggests that the initial binding sites of the two substrates of Enzyme IIlac are separated by at least the distance of the tosyl moiety. Radioactive methyl 6-O-(p-toluenesulfonyl) beta-galactoside was not converted into a phosphorylated product in the reaction mixtures, i.e. it is a true dead-end inhibitor. Inhibition of beta-galactoside transport into whole cells by tosyl galactosides was competitive, with an apparent Ki of 5-10 mM, an order of magnitude higher than the Ki for inhibition of phosphorylation by membrane preparations. This result suggest that a significant level of unphosphorylated phospho-Factor IIIlac is present inside the cells, or that cellular levels of this compound are considerably lower than those used for in vitro sugar phosphorylation assays. Radioactive tosyl galactoside inhibitor was not transported into whole cells.