Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.     

Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174.

In order to examine the possible role of Escherichia coli DNA polymerase II in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded phi X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase II was observed. Furthermore, DNA polymerase II did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II.     

Molecular mechanisms of induced mutagenesis: Replication in vivo of bacteriophage φX174 single-stranded,ultraviolet light-irradiated DNA in intact and irradiated host cells

Genetic analysis has revealed that radiation and many chemical mutagens induce in bacteria an error-prone DNA repair process which is responsible for their mutagenic effect. The biochemical mechanism of this inducible error-prone repair has been studied by analysis of the first round of DNA synthesis on ultraviolet light-irradiated φX174 DNA in both intact and ultraviolet light-irradiated host cells. Intracellular φX174 DNA was extracted, subjected to isopycnic CsCl density-gradient analysis, hydroxylapatite chromatography and digestion by single-strand-specific endonuclease S₁. Ultraviolet light-induced photolesions in viral DNA cause a permanent blockage of DNA synthesis in intact Escherichia coli cells. However, when host cells are irradiated and incubated to fully induce the error-prone repair system, a significant fraction of irradiated φX174 DNA molecules can be fully replicated. Thus, inducible error-prone repair in E. coli is manifested by an increased capacity for DNA synthesis on damaged φX174 DNA. Chloramphenicol (100 μg/ml), which is an inhibitor of the inducible error-prone DNA repair, is also an inhibitor of this particular inducible DNA synthesis.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12

The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication. A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant. Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant. The strain showed no Weigle reactivation. From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant. It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.     

Weigle reactivation of the single-stranded DNA phage f1

Summary The survival of ultraviolet light (UV) damaged single-stranded DNA bacteriophage f1 is increased when the Escherichia coli host is irradiated with UV prior to infection. This repair, called Weigle reactivation, is multiplicity independent and is absent in recA and in lexA mutants. The function of the recA-lexA repair system needed is repair and not recombination, as demonstrated by the absence of Weigle reactivation in mutants that are recombination proficient but defective in repair of double-stranded DNA. Weigle reactivation of f1 requires high levels of the recA protein, and in addition activation of recA or another protein. This activation can be produced by UV irradiation, or by the tif-1 allele of recA together with the spr allele of lexA. Mutagenesis of f1 has the same requirements as W-reactivation, and in addition requires UV irradiation of the phage.     

Inducible repair of thymine ring saturation damage in phi X174 DNA

The susceptibility to inducible SOS repair of 5,6-dihydroxy-dihydrothymine (t') damage in single-stranded phi X174 DNA has been measured. Following exposure to osmium tetroxide, which introduces t' damage in DNA under the conditions used, biological survival of the DNA infected into spheroplasts of Escherichia coli which had received prior exposure to ultraviolet light was higher than in unexposed spheroplasts. From our results, we conclude that approximately 63% of the biological damage from t' products, which is one of the classes of damage present in DNA following ionizing radiation, is susceptible to repair by the inducible SOS repair system.     

Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.     

SOS processing of unique oxidative DNA damages in Escherichia coli

phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.     

SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so.

As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.     

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Novel replicative properties of a capsid mutant of bacteriophage phi chi 174.

A capsid mutant of bacteriophage phi chi 174 demonstrates altered requirements for the conversion of viral single-stranded DNA to double-stranded replicative form DNA. In the presence of puromycin at 42 C, wild-type phi chi 174 is unable to complete this replicative event, whereas phi chi ahb is able to do so. Furthermore, in contrast to wild-type phi chi 174, formation of phi chi ahb parental replicative form DNA is sensitive to rifampin under certain experimental conditions. These data suggest that the mutant capsid proteins of phi chi ahb influence the biosynthesis of phi chi ahb complementary strand DNA.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repair-defective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet light-irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins.     

Studies on the biological role of DNA methylation: III Role in excision of one-genome long single-stranded phi X 174 DNA.

Accumulation of replicative intermediates of the bacteriophage phi X174 was observed in E. coli C infected cells when phage DNA methylation has been inhibited by nicotinamide or when cells were infected with a temperature-sensitive mutant in gene A. Analysis of the accumulating replicative intermediates by electron microscopy revealed that these molecules are composed of double-stranded DNA rings with multiple-genome length single-stranded "tails". These results suggest that the single 5-methylcytosine residue present in the phage DNA serves as a recognition site for the gene A protein mediating the excision of one-genome long phage DNA. This excision process is oligatory for the final maturation of the phage.     

Presence of inducible DNA repair in Bacillus thuringiensis

Weigle reactivation of ultraviolet-irradiated luminal diameter 8 bacteriophage was observed after ultraviolet treatment of Bacillus thuringiensis cells. A slight increased frequency of clear plaque mutants was detected among the survivors. The kinetics of induction of the phage reactivation and phage mutagenesis have been determined. The presence of chloramphenicol before and after irradiation abolished the induction of repair and mutagenesis. These experiments suggest that, in spite of the relatively small mutagenic response in bacteriophage progeny, B. thuringiensis has an inducible repair system responsible to the significant Weigle reactivation of irradiated phage.     

Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069.

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.