Endonuclease from Micrococcus luteus Which Has Activity Toward Ultraviolet-Irradiated Deoxyribonucleic Acid: Its Action on Transforming Deoxyribonucleic Acid

An endonuclease purified from Micrococcus luteus makes single-strand breaks in ultraviolet (UV)-irradiated, native deoxyribonucleic acid (DNA). The purified endonuclease is able to reactivate UV-inactivated transforming DNA of Haemophilus influenzae, especially when the DNA is assayed on a UV-sensitive mutant of H. influenzae. After extensive endonuclease action, there is a loss of transforming DNA when assayed on both UV-sensitive and -resistant cells. The endonuclease does not affect unirradiated DNA. The results indicate that the endonuclease function is involved in the repair of biological damage resulting from UV irradiation and that the UV-sensitive mutant is deficient in this step. We interpret the data as indicating that the various steps in the repair of DNA must be well coordinated if repair is to be effective.     

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 Decrease in Integration of Haemophilus influenzae Transforming Deoxyribonucleic Acid in Sensitive and Resistant Cells

The decrease in integration of transforming deoxyribonucleic acid (DNA) caused by ultraviolet irradiation of the DNA was found to be independent of the presence or absence of excision repair in the recipient cell. Much of the ultraviolet-induced inhibition of integration resulted from the presence in the transforming DNA of pyrimidine dimers, as judged by the photoreactivability of the inhibition with yeast photoreactivating enzyme. The inhibition of integration made only a small contribution to the inactivation of transforming ability of the DNA by ultraviolet radiation.     

Genetic Analysis of Repair of Ultraviolet Damage by Competent and Noncompetent Cells of Bacillus subtilis

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T(-) has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.     

Deoxyribonucleic Acid Polymerase Activity Associated with Strain MC29 Tumor Virus

Chick embryo cells infected with strain MC29 tumor virus yielded progeny virus that contained detectable deoxyribonucleic acid (DNA) polymerase within the first 48 hr after infection. The noninfected culture fluids displayed no such enzyme activity when examined in an identical manner. Enzyme activity was greatly stimulated by adding DNA template to the reaction mixture and required all four deoxyribonucleoside triphosphates for full activity. When calf thymus DNA was used to direct synthesis, the DNA polymerase from the MC29 virus catalyzed the formation of DNA product having a higher buoyant density in CsCl. DNA product formed in the reaction directed by Micrococcus lysodeikticus DNA had the same buoyant density as the template DNA.     

Endonuclease from Micrococcus luteus Which Has Activity Toward Ultraviolet-Irradiated Deoxyribonucleic Acid: Purification and Properties

An endonuclease purified approximately 3,200-fold from Micrococcus luteus is active on native ultraviolet-irradiated deoxyribonucleic acid (DNA), but is inactive on unirradiated native or denatured DNA and has no activity toward irradiated denatured DNA. The major type of lesion for the nucleolytic activity is the cyclobutane pyrimidine dimer. The enzyme makes a number of single-strand breaks approximately equal to the number of dimers, but dimers are not excised. This endonuclease-a small molecular weight protein-therefore has all the attributes hypothesized for the first enzyme in the sequential steps in repair of DNA in vivo. Another paper shows that the endonuclease is able to reactivate ultraviolet-irradiated transforming DNA.     

Integration and Repair of Ultraviolet-Irradiated Transforming Deoxyribonucleic Acid in Haemophilus influenzae

The extent of association between donor transforming deoxyribonucleic acid (DNA) and recipient DNA in Haemophilus influenzae as a function of ultraviolet (UV) dose to the transforming DNA has been measured by isopycnic analysis of lysates of (3)H-labeled recipient cells exposed to DNA labeled with (32)P and heavy isotopes. Except for doses above 15,000 ergs/mm(2), the results of these measurements are in good agreement with previous estimates made by another technique. Experiments with a mutant temperature sensitive for DNA synthesis and another mutant defective in excision of pyrimidine dimers suggest that the discrepancy between the methods of high doses results from DNA synthesis, in which portions of the associated donor DNA containing pyrimidine dimers are excised and broken down, and the components are reutilized for synthesis.Repair of UV-irradiated, transforming DNA during incubation of recipient cells is observed as an increase in transforming ability when fractions from CsCl gradients of cell lysates are assayed on excision-deficient cells. When transforming DNA containing markers of different UV sensitivities is used, repair of the UV-resistant nov marker by excision proficient cells takes place exclusively in the donor DNA that is associated with recipient DNA, and this repair is observed even in the absence of DNA synthesis. However, no repair is observed in the case of the more UV-sensitive str marker, possibly because excision events may remove a large fraction of the integrated str markers in addition to repairing a small fraction of the integrated DNA containing this marker.     

Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans.

Four proteins, alpha beta, gamma, and delta, preferentially synthesized in ultraviolet light-treated cells of Micrococcus radiodurans, were characterized in terms of their molecular weights and isoelectric points. Within the sublethal-dose range, the differential rate of synthesis for these proteins increased linearly with the inducing UV dose. The degree of induction reached 100-fold, and the most abundant protein beta, amounted to approximately 2% of the total newly synthesized protein after irradiation. Damage caused by ionizing radiation or by treatment with mitomycin C also provoked the synthesis of the four proteins. The proportions between the individual proteins, however, varied strikingly with the damaging agent. In contrast to treatments which introduced damage in the cellular deoxyribonucleic acid, the mere arrest of deoxyribonucleic acid replication, caused by nalidixic acid or by starvation for thymine, failed to elicit the synthesis of either protein. Repair of deoxyribonucleic acid damage requires that a number of versatile and efficient processes by employed. It is proposed that the induced proteins participate in deoxyribonucleic acid repair in M. radiodurans. Mechanisms are discussed which would allow a differentiated cellular response to damages of sufficiently distinctive nature.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Postreplication repair in Saccharomyces cerevisiae.

Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light (2 to 4 J/m2). Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were "chased," the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, we concluded that postreplication repair in excision-defective mutants (or leaky mutants) does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.     

Repair of Pyrimidine Dimer Damage Induced in Yeast by Ultraviolet Light

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.     

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.     

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

Cells of Bacillus subtilis recA1 are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system.     

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.     

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Antibacterial nitroacridine, Nitroakridin 3582: effects on bacterial growth and macromolecular biosynthesis in vivo

The antibacterial drug Nitroakridin 3582 inhibited the growth of selected grampositive bacteria more strongly than it inhibited the growth of gram-negative bacilli. Nitroakridin at concentrations of the order of 5 x 10(-5)m induced lysis of Bacillus licheniformis and Micrococcus lysodeikticus. At concentrations less than 10(-4)m, Nitroakridin 3582 reduced the exponential growth rate of Escherichia coli C-2; at 10(-4)m the drug was bacteriostatic, and, at concentrations greater than 10(-4)m, it was bactericidal. Prolonged bacteriostasis resulted in the formation of long filaments by E. coli, Serratia marcescens, Shigella sonnei, and Proteus mirabilis. The reversible effects of Nitroakridin 3582 on the growth of E. coli correlated with partial inhibitions of deoxyribonucleic acid biosynthesis; ribonucleic acid and protein syntheses were inhibited less strongly. Nitroakridin 3582 at concentrations greater than 2 x 10(-4)m, which block deoxyribonucleic acid biosynthesis, produced an accelerated bactericidal action.     

Repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Haemophilus influenzae

Ultraviolet-sensitive and wild-type Haemophilus influenzae cells were exposed to irradiated and unirradiated transforming deoxyribonucleic acid (DNA) containing a marker which can be linked to another marker in the cells. Lysates were made after various times of incubation and assayed for transforming activity on an excisionless recipient. Repair can be noted as an increase in activity from the irradiated donor DNA after its linkage to the recipient DNA. No repair can be observed in a mutant which is unable to integrate transforming DNA. There is a little repair in another mutant which is unable to excise pyrimidine dimers. H. influenzae cells also repair nondimer damage, as judged by the increase in activity observed in lysates made with irradiated and maximally photoreactivated DNA.     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.