Kaney Ebisuzaki

A DNA-dependent ATPase from E.coli infected with bacteriophage T4

A DNA-dependent ATPase has been purified from E.coli infected with bacteriophage T4. The enzyme has a low molecular weight and is not detectable in uninfected E.coli. In the presence of DNA, ATP is cleaved to ADP and inorganic phosphate. Calf thymus DNA, E.coli DNA and heat-denatured T4 and T7 bacteriophage DNAs stimulate the ATPase. However, native T4 and T7 DNAs do not activate the enzyme. In addition to ATP breakdown, the enzyme catalyzes dATP and, to a lesser extent, CTP degradation to their respective diphosphates. No exo- or endonucleolytic activity has been detected. The role of DNA in the reaction is currently under investigation.

Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3‐aminobenzamide, an inhibitor of poly(ADP‐ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP‐ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP‐ribose) DNA strand break DNA repair

Pathways of DNA repair in T4 phage. I. Methyl methanesulfonate sensitive mutant.

Abstract A new T4 phage mutant with increased sensitivity to methyl methanesulfonate (MMS) and ultraviolet (UV) irradiation is described. From a study of the relationship of this mutant to other DNA repair mutants, we have speculated on the pathways of repair of MMS-and UV-damaged T4 phage.

DNA repair-recombination functions in the DNA processing pathway of bacteriophage T4

Abstract It was previously shown that gene 49 codes for an endonuclease which functions in a terminal step in T4 maturation and that gene 49 mutations were suppressed by mutations in uv-sensitive genes, uvs X and uvs Y. Here we have observed that a gene 49 mutation was also suppressed by mutations in genes 46, 47, and 59. Our results suggest that the genes uvs X, uvs Y, 46, 47, and 59 function in a common DNA repair pathway. We suggest that the DNA repair functions constitute part of the DNA processing pathway and that suppression of the gene 49 mutation occurs because the DNA intermediates formed by mutational blocks in genes 46, 47 59, uvs X, and uvs Y bypass the gene 49 function and reenter the DNA processing pathway just prior to the genes 16–17 function(s).

Polynucleotide kinase mutant of bacteriophage T4

SummaryWe report the isolation and biological properties of two T4 phage mutants which are deficient in polynucleotide kinase. These two mutants are wild type with respect to replication, genetic recombination and DNA repair (as measured by sensitivity to UV irradiation, γ-irradiation and 32P decay). These negative results suggest that this phage-induced enzyme is not a critical factor in DNA metabolism.

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.

Intergenic suppression of amber polynucleotide ligase mutation in bacteriophage T4. II

Abstract An intergenic suppressor ( m ) of the amber ligase mutation ( amH 39 x ) was isolated and a number of possible mechanisms of suppression were investigated. The intergenic suppressor does not appear to involve suppression of the amber codon. Biological tests and enzyme assays indicated that the intergenic suppressor mutation m is not associated with a nuclease defect. Complementation studies revealed incomplete dominance of both alleles ( m or m + ) of the suppressor gene, suggesting a noncatalytic function for the wild-type suppressor gene ( m + ). In a mixed infection of E. coli B with an amber ligase mutant and the intergenic suppressed strain the former was preferentially excluded. The m mutants are rapid lysis mutants but they differ from the rII mutants in a number of properties.

Inhibitors of Poly(ADP-Ribose) Polymerase Prevent Friend Cell Differentiation

Friend et al. [1] made the striking observation that dimethyl sulfoxide (DMSO)-treated Friend erythroleukemia cells (FEL) differentiate in vitro. Subsequently, a number of other chemicals, including butyric acid, hypoxanthine and hexamethylene bis-acetamide were shown to induce Friend cells [2]. These inducers appear to remove a block in the differentiation process but the mechanisms involved are unknown. The finding that benzamide and nicotinamide induced Friend cells [3, 4] suggested that since both compounds were inhibitors of poly(ADP-ribose) polymerase, poly(ADP-ribosylation) may have a role in the differentiation process. Furthermore, since poly(ADP-ribose) polymerase requires DNA strand breaks for activity [5], these observations implicated DNA strand breaks in FEL differentiation.

The coupling of DNA repair-recombination functions with DNA replication in bacteriophage T4: a new DNA repair mutant

The requirement of DNA repair-recombination functions for T4 phage DNA replication has been known for some time but the underlying basis for this relationship has been unclear. This report is concerned with a new uv-sensitive gene [uvsU], whose function appears to bridge these two major activities of DNA. The [uvsU] mutant fails to complement [uvsX] mutants but uvsU maps in a region distinct from uvsX. Furthermore, the uvsU mutation specifically suppressed the DNA replication defect but not the uv sensitivity of the uvsX mutation. The previously discovered uvsW gene, whose mutations suppress the DNA replication defects of gene 59, 46, and 47 mutations, seems to have an analogous role. As a possible explanation for these observations, it is suggested that the uvsW and uvsU gene products (gps) couple the DNA repair-recombination and replication functions by controlling the entry of DNA intermediates from the replication pool into the DNA repair-recombination pathway. Furthermore the suppression data are interpreted to suggest that the gps uvsW, 59, 46, and 47 function together. Similarly the gps uvsU and uvsX may form a functional unit.

A new suppressor of mutations in the DNA repair-recombination genes of bacteriophage T4: sur.

A new mutation designated sur was isolated as a suppressor of a mutation in the uvsX gene of T4 phage. Unlike the other suppressors of mutations in genes involved in DNA repair and recombination, sur has a wide range, suppressing both DNA repair and replication defects in mutations in genes uvsX, uvsY, 46, 47, and 59. However, its suppressor functions may be confined to the uvsX-uvsY DNA repair pathway since sur did not suppress a mutation in the denV gene. The sur mutation results in an increased degradation of host DNA to an acid-soluble form, but this increase was blocked by a mutation in gene 46 (nuclease) indicating that the sur function is involved in an earlier step in the degradation of host DNA. This increased degradation of host DNA might be a reflection of a compensatory increase in an alternate DNA repair activity in the [sur] mutant.