Efficient Excision Research Articles

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda: Integration and excision involve distinct interactions of integrase with arm-type sites

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome ( attP) and the Escherichia coli genome ( attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int “arm-type” binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm type sites.

Cell cycle effects on the basal and DNA-damaging-agent-stimulated ADPRT activity in cultured mammalian cells

ADP-ribosyl transferase (ADPRT) is a DNA-dependent chromatin-associated enzyme which covalently attaches ADP-ribose moieties derived from NAD + to protein acceptors to form poly(ADP-ribose). ADPRT activity is strongly stimulated by breaks in DNA, and it is suggested that its activity is required for efficient DNA excision repair. In this paper, a cell-cycle-dependent fluctuation of basal ADPRT activity was demonstrated by measuring it in permeabilized FL cells. The cell used was subjected to arginine starvation for 48 h before being released from the block by replacement of deficient medium with complete medium and cells in different proliferating stages were traced by [ 3H]TdR pulse labelling and obtained at different intervals after block release. The peak basal ADPRT activity appeared 4–6 h after the appearance of the peak of DNA synthesis. After treating the cells with MNNG (10 −4 M), MMS (10 −3−10 −4M) and 4NQO (10 −5 M) for 90 min just after release of the block, the ADPRT activity was markedly stimulated. It was further demonstrated that the effects of MNNG/4NQO and cell cycle influence on the level of poly(ADP-ribose) synthesis appear to be additive. While concerning MMS, quite a different pattern of ADPRT stimulation in the cell cycle was demonstrated, i.e., the activity of ADPRT stimulation of 10 −3 M MMS was found to be completely dependent on the basal ADPRT activity. In the cells with the highest basal ADPRT activity 12 h after block release, the MMS-induced ADPRT stimulation could not be observed. It was suggested that more than one pathway might be present in ADPRT stimulation induced by DNA-damaging chemicals, and the cells synchronized in late G 1 stage might be the most suitable for demonstrating poly(ADP-ribose) synthesis after DNA damage.

Excision of pyrimidine dimers from the DNA of Neurospora.

Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmunoassay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers. UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency.

Repair replication in ultraviolet light-irradiated protoplasts of Daucus carota

Colony-forming ability and DNA replication in protoplasts isolated from suspension cultures of the wild carrot Daucus carota were found to be moderately resistant to ultraviolet irradiation. 37% survival was achieved after a dose of 100 J/m2, while 50% inhibition of replication occurred at a dose of 80 J/m2. Analysis of the thymine dimer content of DNA from irradiated protoplasts after dark incubation confirmed the existence of an efficient excision repair mechanism. A maximum rate of excision of 25 000 dimers/cell per h was achieved at a dose of 100 J/m2. A novel method for inhibiting DNA replication with the fungal metabolite compactin allowed the quantitation of repair replication in protoplasts isolated from dividing cells by chromatography of the DNA on BND-cellulose. Ultraviolet light-stimulated incorporation of [3H]thymidine was demonstrated and a patch size of 70 nucleotides calculated. The properties of the excision repair mechanism of Daucus protoplasts are therefore similar to those of mammalian systems. However, excision alone is insufficient to account for the removal of all dimers induced by natural sunlight.

ADP-ribosyl transferase activity in Trypanosoma brucei

Nuclear adenosine diphosphoribosyl transferase (ADPRT) catalyses the covalent modification of chromatin proteins by (ADP-ribose) n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity.

The stage specific inhibition of Dictyostelium discoideum spore germination by the mutagen 4-nitroquinoline 1-oxide

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 μg/ml (26 μM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O 2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [ 3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of β-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.

DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation.

The nuclear enzyme ADP-ribosyl transferase (ADPRT) catalyses the formation of poly(ADP-ribose)-modified chromatin proteins from NAD+ (refs 1–5) and is entirely dependent on DNA6 containing nicks7–11. Nuclear ADPRT activity is required for efficient DNA excision repair12,13, probably because it regulates DNA ligase activity14. Indirect evidence has suggested that ADPRT activity may also be involved in control of gene expression and cell differentiation15–21. We report here an obligatory involvement of ADPRT activity in the differentiation of muscle cells. Inhibitors of ADPRT activity reversibly inhibit both fusion of myoblasts to form multi-nucleate muscle fibres and the differentiation-specific increase in creatine phos-phokinase (CPK) activity. These two markers of differentiation can also be reversibly inhibited by depriving the cells of nicotinamide and thus lowering their cellular NAD content. Specific gene expression sometimes requires gene rearrangements or DNA transposition; this implies that DNA strand-breaking and rejoining might be involved in gene expression. We also describe the appearance during cytodifferentiation of single-strand DNA breaks which are not due to a general deficiency in DNA repair.

Differentiation of the mammary gland and susceptibility to carcinogenesis.

It has been demonstrated that in humans certain factors such as early menarche, late pregnancy, and nulliparity are associated with a higher risk of developing breast cancer, while early pregnancy acts as a protective factor. Induction of mammary cancer in rats by administration of the chemical carcinogen 7,12-dimethylbenz(a)anthracene reveals that the same factors influencing human breast cancer risk also affect the susceptibility of the rat mammary gland to the chemical carcinogen. Nulliparous rats and rats undergoing pregnancy interruption are more susceptible to developing carcinomas. This fact has been attributed to the incomplete differentiation of the gland at the time of carcinogen administration. Parous rats are resistant to the carcinogenic effect of DMBA, which is explained by the complete development of the gland attained during pregnancy and lactation. This development is manifested by the differentiation of terminal end buds into secretory units, which have a smaller proliferative compartment; the epithelial cells of these secretory units have a longer cell cycle, less avidity for binding DMBA, and possess a more efficient DNA excision repair capacity.

Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5.

The excision of Tn5 from sites of insertion in the Escherichia coli genome was studied by examining the reversion of lac::Tn5 insertion mutations to lac+. We find that: (i) the frequency of excision depends on the site of Tn5 insertion, (ii) excision occurs efficiently in recA- cells, (iii) excision does not require a Tn5-encoded transposition function, and (iv) efficient excision requires the inverted repeats of Tn5. We propose that excision of Tn5 is similar to the formation of spontaneous deletions and occurs by slippage during DNA synthesis.

Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis.

The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites. Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight. In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells. Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands.

Unprogrammierte DNA-Synthese (DNA-Reparatur) in Milz-und Thymuszellen der Ratte nach Einwirkung von UV-Licht, Röntgenstrahlen oder Methylmethansulfonat in vitro / Unscheduled DNA Synthesis (DNA-Repair) within Splenic and Thymic Cells of the Rat under the Influence of UV-Light, X-Irradiation and/or Methyl-methanesulfonate

Unscheduled DNA synthesis (UDS) of splenic and thymic cells of the rat has been stimulated in vitro by UV-light (8-128 J x m-2), X-rays (120-3480 rd), methyl-methanesulfonate (MMS), and/or a combination of UV-light and X-irradiation. The height of UDS-induced stimulation of incorporation of [3H]thymidine into splenic and thymic cell DNA at saturation doses of UV-light (splenic cells: 8, thymic cells: 96 J x m-2) or X-irradiation (splenic cells: 960, thymic cells: approximately 3480 rd) suggest that the greater sensitivity of T-cells (represented by thymic cells) towards UV-light and the greater sensitivity of B-cells (represented by splenic cells) towards X-rays can be explained-at least partly-in terms of less efficient excision repair systems.

The role of dimer excision in liquid-holding recovery of UV-irradiated haploid yeast

We have directly tested the theory that liquid-holding recovery is due to an increase in the efficiency of excision-repair during holding in non-growth conditions, by assaying the dimers present in UV-irradiated cells held in saline, in growth medium or first in saline then in growth medium. We observed no differences in the amount of excision in any conditions. By assaying the kinetics of excision and comparing that with the timing of DNA synthesis, we have tested the theory that holding in growth medium allows more repair by extending the time available for it. We found that the observations were more consistent with the onset of DNA synthesis being dependent on the amount of repair rather than the converse. We have analysed the role of repair in liquid- holding recovery in a series of split-dose experiments. As Parry and Parry found, yeast cells which have been irradiated and held in non-growth conditions were much more resistant to further UV-irradiation. The increase in resistance was proportional both to the degree of fractionation of the dose and to the size of the first dose. No effect was observed if this was below 30 J β m −2. We found that the cells were able to excise more of the dimers induced if the UV dose was fractionated. We have shown that part of this increase in efficiency of excision is due to the relief of “dimer interference”. “Dimer interference” is the name given to the inhibition of excision of a dimer by the presence of a neighbouring dimer. Most of the increase in efficiency, however, was due to the induction of more efficient excision repair per se, that is the excision of a greater fraction of the dimers present than could be excised in uninduced cells. Among the incidental observations we have made which are new and likely to be of interest are (1) that stationary phase cells showed a lag in the onset of excision, but log phase cells did not; (2) that excision was nevertheless constitutive in that it occurred in the presence of the concentrations of cycloheximide inhibitory to protein synthesis and (3) that caffeine affected but did not inhibit dimer excision.

DNA sequence of the int-xis-Pi region of the bacteriophage lambda; overlap of the int and xis genes.

The DNA sequence of the int and xis genes of lambda bacteriophage, as well as that of the PI promoter and a region of unknown function beyond this, was determined by the chain termination procedure. The Pribnow box sequence of the PI promoter lies just within the xis gene, and both possible sites of mRNA initiation from PI are within the xis gene. The end of the xis gene in its turn overlaps the start of the int gene by 23 base pairs, in a different reading frame. This overlap may play a role in ensuring efficient excision of the prophage in response to natural induction signals.

Incidence and complications of ureteral stump pathology.

The authors report on 58 patients with ureteral stump disease. Therapeutic errors, certain specific conditions, but also nephrectomies done for diseases other than pyelo-calyceal tumors and tuberculosis may increase the severity of ureteral stump pathology. The clinical symptoms are often vague, and are demonstrated too late. The incidence may be reduced and the outcome improved by removal of the ureter whenever favourable conditions for development exist, and by looking for them by efficient exploration and complete excision, if nephrectomy is followed by even less marked changes.

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.

Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism.

Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.