Photoenzymatic Repair of Ultraviolet Damage in DNA : I. Kinetics of the reaction

As previously reported, ultraviolet-inactivated bacterial transforming DNA can be restored to activity by an enzyme-like agent from bakers' yeast which requires light for its activity. Kinetics of this reaction, in the presence and absence of inhibitors, are found consistent with the Michaelis-Menten reaction scheme, with the sites of ultraviolet damage on the DNA serving as substrate and the repaired structure as product. Kinetic studies with different light intensities suggest that the necessary illumination causes photolysis of the enzyme-substrate complex with concurrent repair of the DNA. Competitive inhibition of irradiated transforming DNA repair, which occurs when irradiated non-transforming DNA is present in the same reaction mixture, permits ultraviolet damage (of the kind capable of being photoreactivated) to be detected in any type of DNA.     

Mechanism of Inactivation of Haemophilus influenzae Transforming Deoxyribonucleic Acid by Sonic Radiation

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to sonic radiation of various durations. Reductions in transforming ability of the DNA, cellular DNA uptake, and integration into the genome, and single- and double-stranded molecular weights of the transforming DNA were measured and compared. We conclude that (i) sonic radiation causes DNA strand breaks (almost always double-strand breaks with relatively few alkaline-labile bonds), the number increasing with exposure until the double-stranded molecular weight is reduced to less than 10(6) daltons; and (ii) since transformation is reduced about as much as integration and much more than uptake, inactivation of transforming DNA by sonic radiation appears to be caused mostly by failure of Haemophilus cells to integrate the transforming DNA that is taken into the cells. These results are similar to those for inactivation by X radiation but differ from those for ultraviolet radiation. A strand break caused by sonic radiation, however, does not necessarily inactivate the transforming DNA, whereas in the case of ionizing radiation it may. The results may be fit by the model proposed by Cato and Guild. From our data and the equation of Lacks, the minimum active site of DNA necessary for transformation and the frequency of exchanges between donor and recipient strands upon integration of transforming DNA were estimated as 0.35 x 10(6) to 0.7 x 10(6) daltons and 0.15 to 0.4 switches per 10(6) daltons, respectively.     

The effect of chlorpromazine on transformation in Bacillus subtilis

Summary In the presence of the widely used tranquilizer, chlorpromazine, transforming DNA of Bacillus subtilis is photoinactivated by long-wave ultraviolet light. The loss of biological activity is predominantly caused by lack of binding of the DNA to recipient cells and the introduction of single-strand breaks in the treated DNA.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Killing of Haemophilus influenzae Cells by Integrated Ultraviolet-Induced Lesions from Transforming Deoxyribonucleic Acid

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.     

Reovirus: Effect of Noninfective Viral Components on Cellular Deoxyribonucleic Acid Synthesis

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.     

In vitro repair of UV-or X-irradiated bacteriophage T4 DNA by extract from blue-green alga Anacystis nidulans

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.Abbreviations UV ultraviolet - TA transforming activity - PN-ligase polynucleotide ligase - NAD nicotinamide adenine dinucleotide - dNTP deoxynucleoside triphosphates - dATP, dGTP, dTTP triphosphates of deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine, respectively     

Repair of Irradiated Transforming Deoxyribonucleic Acid in Wild Type and a Radiation-Sensitive Mutant of Micrococcus radiodurans

The survival of biological activity in irradiated transforming deoxyribonucleic acid (DNA) has been assayed in the wild type and a radiation-sensitive mutant of Micrococcus radiodurans. The frequency of transformation with unirradiated DNA was lower in the mutant to about the same extent as the mutant's increased sensitivity to radiation. However, in both the wild type and the mutant, the irradiated DNA that was incorporated into the bacterial genome was repaired to the same extent as determined by the loss of transforming activity with increasing radiation dose. This applied to DNA irradiated either with ionizing or ultraviolet (UV) radiation. The rate of inactivation of biological activity after UV radiation was the same in any of the DNA preparations tested. For ionizing radiation, the rate of inactivation varied up to 40-fold, depending on the DNA preparation used, but for any one preparation was the same whether assayed in the wild type or the radiation-sensitive mutant. When recipient bacteria were irradiated with ionizing or UV radiation immediately before transformation, the frequency of transformation with unirradiated DNA fell, rapidly and exponentially in the case of the sensitive mutant but in a more complicated fashion in the wild type. The repair of DNA irradiated with ionizing radiation was approximately the same whether assayed in unirradiated or irradiated hosts. Thus, irradiation of the host reduced the integration of DNA but not its repair.     

Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II. Effect of inhibitors and DNA precursors.

Despite their great sensitivity to ultraviolet light purified human B and T lymphocytes are capable of complete repair provided that the ultraviolet dose does not exceed 0.5 Jm-2. Their capacity to repair, as measured by the restoration of DNA supercoiling in preparations of nucleoids, and their survival are significantly increased in the presence of deoxyribonucleosides. Certain agents which inhibit semi-conservative DNA synthesis (hydroxyurea, 1-beta-D-arabinofuranosylcytosine (arafCyt) either stop or delay the repair process in lymphocytes. The effect of hydroxyurea is eventually overcome spontaneously, but changes in the sedimentation behaviour of ultraviolet-irradiated nucleoids caused by arafCyt can only be neutralized by addition of deoxycytidine. The effective inhibition of repair by arafCyt permits the detection of extremely small amounts of ultraviolet damage and also the estimation of when repair is complete.     

Efficiency of Escherichia coli repair processes on uv-damaged transforming plasmid DNA

A comparison has been made of the efficiencies with which the dark repair processes of Escherichia coli act on ultraviolet irradiated bacterial chromosomal DNA and ultraviolet damaged transforming plasmid DNA. It is shown that postreplicational repair pathways act very inefficiently on transforming plasmid DNA, and that the majority of repair is carried out by excision repair pathways. However, even excision repair pathways act less efficiently on damaged plasmid DNA than they do on chromosomal DNA. The large effect of mutations in recB on plasmid survival suggests that the product of this gene may be essential for the excision repair pathways which act on plasmid DNA, but not for those which act on chromosomal DNA.     

Integration Efficiency in DNA-Induced Transformation of Pneumococcus. I. a Method of Transformation in Solid Medium and Its Use for Isolation of Transformation-Deficient and Recombination-Modified Mutants

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.     

Integration Efficiency in DNA-Induced Transformation of PNEUMOCOCCUS. II. Genetic Studies of Mutant Integrating All the Markers with a High Efficiency

Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Effect of transforming DNA on growth and frequency of mutation of Streptococcus pneumoniae.

We studied the effect of the presence of homologous transforming DNA on the growth of several transformable strains of Streptococcus pneumoniae and on the frequency of mutation of these strains to various antibiotic resistances. We observed no effect on growth until the strains became competent, when growth was depressed. At the end of the competence period, some strains showed recovery to varying degrees, whereas others showed evidence of cell death. Growth was also depressed by the presence of DNA from Escherichia coli, indicating that recombination was not likely to be the cause of the observed effect. Furthermore, cell death was not caused by the induction of a prophage. Several of the strains showed increased mutation frequencies during the competence period, although treatment with E. coli DNA gave no such effect, indicating that the mutagenesis was due to recombination. We observed no mutagenesis due to UV irradiation of the strains. The possibility that integration of the transforming DNA may produce lesions which induce error-prone repair is discussed. Furthermore, a strain that showed no mutability by transforming DNA, indicating the presence of a more efficient repair system, gave evidence of producing higher amounts of the hex system when competent, and the possible relationship between these properties is discussed.     

Regulation of DNA replication by ATR: signaling in response to DNA intermediates

The nuclear protein kinase ATR controls S-phase progression in response to DNA damage and replication fork stalling, including damage caused by ultraviolet irradiation, hyperoxia, and replication inhibitors like aphidicolin and hydroxyurea. ATR activation and substrate specificity require the presence of adapter and mediator molecules, ultimately resulting in the downstream inhibition of the S-phase kinases that function to initiate DNA replication at origins of replication. The data reviewed strongly support the hypothesis that ATR is activated in response to persistent RPA-bound single-stranded DNA, a common intermediate of unstressed and damaged DNA replication and metabolism.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.