Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

Split-dose recovery is due to the repair of DNA double-strand breaks

DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad 54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break.     

Neutral sucrose gradient sedimentation of very large DNA from Bacillus subtilis. II. Double-strand breaks formed by gamma ray irradiation of the cells

A procedure has been developed whereby essentially all the DNA from Bacillus subtilis cells can be reproducibly extracted in a form which sediments 2.3 times faster than bacteriophage T2 DNA in a neutral sucrose gradient spun at 20,000 revs/ min. When the cells are irradiated with low (3 to 34 kilorads) gamma ray doses, some DNA moves in a slower peak, which from the previous paper (Levin &; Hutchinson, 1973) appears to be linear DNA. Some of the DNA also sediments ahead of the unirradiated DNA. On incubation of the cells at 37°C under conditions such that single-strand DNA breaks are repaired, the fast-sedimenting component is partially restored, with the DNA sedimenting ahead of it usually disappearing, and the quantity in the slower component decreasing. With 80 minutes incubation the fraction of DNA after various radiation doses in the fast-sedimenting component is the same as the fraction of cells able to form colonies, suggesting that the destruction of the component is responsible for the effect of gamma rays on the ability of a cell to replicate. Single-strand breaks introduced into the DNA within the cells do not affect the fast-sedimenting component, so radiation-induced single-strand breaks are not responsible for the effect of gamma rays on replication.The double-strand break rate for DNA in the cells is 0.010 breaks per mass the size of T2 DNA per kilorad. The fast-sedimenting component in irradiated cells which have not been incubated disappears at a rate equal to one radiationinduced double-strand break formed per genome. Since the fast-sedimenting component in solution is also destroyed by one double-strand break per genome (Levin &; Hutchinson, 1973), it is suggested that this component is the genome in the form of a circle. The correspondence between DNA in the fast-sedimenting form after incubation and the ability of cells to form colonies then indicates that a genome can replicate only if all double-strand breaks are repaired.     

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.     

Double-strand DNA break formation mediated by flap endonuclease-1

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.     

Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H₂O₂, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN + HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN + HP-induced fragmentation only ∼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ∼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.     

The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1.9 +/- 0.2, whereas the o.e.r.-value for DNA double-strand breakage was 3.0 +/- 0.1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0.74 x 10(-11) double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0.24 x 10(-11) double-strand breaks per g/mol per Gy under nitrogen.     

Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid.

The structures of the membrane-free nucleoid of Escherichia coli K-12 and of unfolded chromosomal deoxyribonucleic acid (DNA) were investigated by low-speed sedimentation on neutral sucrose gradients after irradiation with 60Co gamma rays. Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA packaging in the bacterial chromosome. The number of domains of supercoiling was estimated to be approximately 180 per genome equivalent of DNA, based on measurements of relaxation caused by single-strand break formation in folded chromosomes gamma irradiated in vivo and in vitro. Similar estimates based on the target size of ribonucleic acid molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of ribonucleic acid single-strand breaks at doses of up to 10 krad; this was born out by experimental measurements. Unfolding of the nucleoid in vitro by limit digestion with ribonuclease or by heating at 70 degrees C resulted in DNA complexes with sedimentation coefficients of 1,030 +/- 59S and 625 +/- 15S, respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing two to three genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma irradiated in vitro. Break formation was linear with doses up to 10 krad and occurred at a rate of 0.27 double-strand break per krad per genome equivalent of DNA (1,080 eV/double-strand break). The influence of possible nonlinear DNA conformations on these values is discussed.     

Repair of double-strand breaks by incorporation of a molecule of homologous DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules ('donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending approximately 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [32P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.     

A method of calculating initial DNA strand breakage following the decay of incorporated 125I

Two sources of individual Auger electron spectra and an electron track code were used with a simple model of the DNA to successfully simulate the single-strand DNA breakage measured by Martin and Haseltine (1981). The conditions of the calculation were then extended to examine patterns of single-strand breaks in both strands of the DNA duplex to score double-strand breaks. The occurrences of five types of break were scored. The total number of double-strand breaks (dsb) per decay at the site of the decay was 0.90 and 0.65 for the different Auger electron spectra. It was shown that for mammalian cells an additional source of double-strand breaks from low LET radiation added approximately 0.17 dsb/decay to each, giving a final total of 1.07 and 0.85 dsb/decay for mammalian cells depending on the electron spectrum. Further is is shown that the energy deposition in the DNA from the iodine decay is very complex, with a broad range of energy depositions and products. Even for a particular energy deposited in the DNA different types of strand break are produced. These are identified and their probabilities calculated.     

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system.

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.     

The Rad51 and Dmc1 recombinases: a non-identical twin relationship

A double-strand break in genomic DNA that remains unrepaired can be lethal for a cell. Indeed, the integrity of the genome is paramount for survival. It is therefore surprising that some cells deliberately introduce double-strand breaks at certain times during their life cycle. Why might they do this? What are the benefits? How are these breaks repaired? The answers to these questions lie in understanding the basis of meiotic recombination, the process that leads to genetic variation. This review summarizes the key roles played by the two recombinases, Dmc1 and Rad51, in the faithful repair of DNA breaks.     

Interpretation of Sucrose Gradient Sedimentation Pattern of Deoxyribonucleic Acid Fragments Resulting from Random Breaks

Mass distribution in a sucrose gradient of deoxyribonucleic acid (DNA) fragments arising as a result of random breaks is predicted by analytical means from which computer evaluations are plotted. The analytical results are compared with the results of verifying experiments: (i) a Monte Carlo computer experiment in which simulated molecules of DNA were individuals of unit length subjected to random "breaks" applied by a random number generator, and (ii) an in vitro experiment in which molecules of T4 DNA, highly labeled with (32)P, were stored in liquid nitrogen for variable periods of time during which a precisely known number of (32)P atoms decayed, causing single-stranded breaks. The distribution of sizes of the resulting fragments was measured in an alkaline sucrose gradient. The profiles obtained in this fashion were compared with the mathematical predictions. Both experiments agree with the analytical approach and thus permit the use of the graphs obtained from the latter as a means of determining the average number of random breaks in DNA from distributions obtained experimentally in a sucrose gradient. An example of the application of this procedure to a previously unresolved problem is provided in the case of DNA from ultraviolet-irradiated phage which undergoes a dose-dependent intracellular breakdown. The relationship between the number of lethal hits and the number of single-stranded breaks was not previously established. A comparison of the calculated number of nicks per strand of DNA with the known dose in phage-lethal hits reveals a relationship closely approximating one lethal hit to one single-stranded break.     

Potentiation of sulphur mustard or cisplatin-induced toxicity by caffeine in Chinese hamster cells correlates with formation of DNA double-strand breaks during replication on a damaged template

Caffeine inhibited the elongation of nascent DNA and induced breaks in the template DNA of sulphur mustard-treated Chinese hamster cells. The sizes of template and nascent DNAs, as indicated by alkaline sucrose gradient sedimentation, were similar suggesting that incision of template DNA occurred opposite gaps formed in nascent DNA by the action of caffeine, forming, effectively, double-strand breaks in DNA. Double-strand break formation was demonstrated, by means of elution of labelled DNA through polycarbonate filters at neutral pH, in both sulphur mustard- and cisplatin-treated cells when they were incubated in the presence of caffeine for 24 h. Double-strand breaks were only formed in that DNA which had been replicated in the presence of caffeine after treatment with sulphur mustard or cisplatin. Non-toxic concentrations of cycloheximide abolished the potentiation by caffeine of sulphur mustard-induced toxicity to Chinese hamster cells and at the same time abolished the formation of the low molecular weight nascent DNA, and as a consequence of its inhibitory effect on DNA synthesis, and the formation of double-strand breaks in DNA. Potentiation of the lethal and clastogenic effects of genotoxic agents by caffeine is therefore due to effects on the rate and mode of DNA synthesis which lead finally to double-strand breaks in DNA.     

Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.     

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.     

In Vitro Repair of Gaps in Bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs

A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-strand breaks revealed that they are all characterized by staggered two-nucleotide long 5' overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5' extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.     

The role of specific DNA base damages in the X-ray-induced inactivation of bacteriophage PM2

Two types of X-ray-induced base damages, alkali-labile sites and thymine ring saturation products, were quantitated in PM2 DNA irradiated in the phage capsid under oxic and anoxic conditions. The extent of formation of these base damages was compared with the number of single- and double-strand breaks and lethal hits produced under the same conditions. The individual inactivation efficiencies of alkali-labile sites and thymine ring saturation products were determined by selectively inducing each of these damages in isolated PM2 DNA by chemical means in vitro and determining the rate of biological inactivation of the treated DNA by transfection. For each lethal X-ray hit induced in oxic conditions there were 1.06 alkali-labile sites, 0.40 thymine ring saturation products, 2.09 singe-strand breaks and 0.11 double-strand breaks in the PM2 genome. In anoxic conditions, the respective number of lesions was 1.00, 0.19, 1.73 and 0.09. The individual inactivation efficiencies of thymine ring saturation products and alkali-labile sites were found to be essentially equal, 7-8 lesions per lethal event in the PM2 genome. Alkali-labile sites and thymine ring saturation products together accounted for 15-20% of the biological inactivation of X-irradiated bacteriophage PM2. The presence or absence of oxygen during irradiation did not affect the contribution to inactivation made by alkali-labile sites, but the contribution by thymine ring saturation products to inactivation was about 2-fold higher in oxic compared with anoxic conditions. With the 4 lesions measured, we have accounted for some 28-34% of the lethal events in X-irradiated PM2 phage, most of the remaining events being caused by as yet unidentified base damages.