Mutation Research, 105 (1982) 19-22
19
Elsevier Biomedical Press
Heat mutagenesis of bacteriophage SOS-induced bacteria*
X174 in
Roeland M. Schaaper and Lawrence A. Loeb** The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted l0 May 1982)
Heat has long been recognized as an ubiquitous environmental mutagen, presumably based on its DNA-damaging properties. Studies with bacteriophage T4 have identified in this respect at least 2 types of heat-induced alterations in DNA, deamination of cytosine causing C ~ T transitions (Baltz et al., 1976) and - less firmly established - the rearrangement of guanine to neoguanine, causing G-~C transversions (Bingham et al., 1976). We have recently evaluated the importance of ~ipurinic sites as an intermediate in mutagenesis. Rates of depurination at physiological conditions are considerable, and increase rapidly at higher temperatures (Lindahl and Nyberg, 1972). Apurinic sites introduced in homopolymeric or natural DNA lead to errors in DNA replication when copied by purified DNA polymerases in vitro (Shearman and Loeb, 1979; Kunkel et al., 1981). In vivo mutagenesis was demonstrated by transfection of depurinated bacteriophage ~X174 viral DNA into 'SOS-induced' E. coli spheroplasts. A large increase in reversion frequency to wild-type was observed when DNA of thX174 amber mutants was used. Mutagenesis was dependent upon preirradiation of the bacteria with ultraviolet light prior to their conversion to spheroplasts and proportional to the number of apurinic sites. Mutagenesis was abolished by treating the depurinated DNA with alkali (Schaaper and Loeb, 1981) or by incubation with an apurinic endonuclease (Schaaper, unpublished results). These results suggest the possible importance of the interaction of apurinic sites with inducible error-prone-repair processes like the E. coli SOS-system (Radman, 1974; Witkin, 1976). In this communication we report an analysis of the effect of heat on intact bacteriophage. The assumption is made that as in the case of naked DNA treated in vitro, the genetic damage includes depurination. Experiments with whole phage however, might provide an additional and more sensitive system for studying depurination and the mechanism of heat mutagenesis. Transfection systems, * This paper replaces an earlier version, in which the wrong Fig. 1 was inserted (Mutation Res., 104 (1982) 75-78). ** To w h o m reprint requests should be addressed.
20 although they are elegant and allow for extensive manipulation of the DNA in vitro, generally suffer from a reduced sensitivity. Large amounts of DNA are required to overcome both the low efficiency of transfection (typically 10 - 4 infective centers per DNA molecule) and the reduction in infectivity o f the DNA as a result of treatment. Thus, studies with intact phage are less variable and are 4 orders o f magnitude more sensitive than those with isolated DNA. ~X174 wild type and am3 phage were heated at 50°C for increasing periods of time in a 10 mM Na citrate, 100 mM KCI buffer, pH 4.50. These acidic conditions were chosen to favor depurination. E. coli C was grown in L-broth to a density of 3 × 108 cells per ml and used as such, or irradiated with 80 J m - 2 UV light (254 nm) and incubated for an additional 45 min at 37°C to allow SOS expression. Appropriate dilutions of amber phage were plated on both kinds of bacteria to determine the number of revertants. Permissive titers were scored by plating on strain HF4714 (sul÷). Differences in survival due to Weigle reactivation were checked by plating identically treated wild-type phage on each of the 3 types of bacteria. The results are shown in Figs. 1 and 2. Inactivation of both wild-type and am3 phage follows a biphasic pattern, in which a rapid initial inactivation is followed by a slower phase. This pattern was confirmed in several other experiments specifically aimed at obtaining accurate survival curves (results not shown). These results are similar to those found by Bleichrodt and van Abkoude (1967), who postulated that in phage preparations, 2 types of phage exist with different thermosensitivities. Depending on its history, each preparation may have different proportions of the 2 phage forms. Apparently, this is the case in Fig. 1, where am3 has a much higher proportion of temperature-resistant phage than the wild-type preparation. After the initial 20 min, both kinds of phage stocks inactivate at similar rates, as judged from the parallel nature of the 2 inactivation curves. The survival of wild-type phage was identical in all 3 strains used and thus no Weigle reactivation is observed on the preirradiated host. This is in agreement with experiments in which depurinated phage DNA was studied by transfection in pre-irradiated spheroplasts (Schaaper and Loeb, 1981). Fig. 2 shows that heat treatment of the phage is only weakly mutagenic in normal hosts. In bacteria preirradiated with UV, however, the reversion frequency is increased nearly 100-fold. Apparently lesions introduced during the heat/acid treatment are refractory to mutagenesis during normal DNA replication, but become highly mutagenic under SOS conditions. In the experiments described above, conditions were chosen such that substantial depurination of the DNA is to be expected. The second, slower part of the inactivation curve (Fig. 1) suggests that during this phase lethal hits are introduced at a rate of about 1 for every 20 min of incubation. We have previously shown (Schaaper and Loeb, 1981) that apurinic sites in ~X174 DNA are predominantly lethal hits and that one site per circle is sufficient to inactivate the molecule. Mutagenesis results from a relatively rare event in which replication presumably
21
,o-,] ,, _
\ 10 -4
,,
I0 z
o')
i0-5
+x w t ~ I0"
'°o
2'o
5'o
,do
Heat treatment (rnin)
'°"o
2'0
|
Heat treatment (min)
Fig. I. Inactivation of ~X174 bacteriophage u p o n various exposures to 50°C at pH 4 . 5 0 : a m 3 was plated on HF4714 (sul + ) ( O ) and wild type (wt)was plated on HF4714 (sul +) (0), E. coil C S O S - (~) and SOS ÷ ( I ) cells (pre-irradiated with 80 J m - 2). Fig. 2. Reversion frequency of ~X174 am3 phage, exposed to 50°C at pH 4.50 for indicated times and plated on E. coli C SOS - ( [] ) and SOS ÷ ( • ) cells.
proceeds past apurinic sites. The exact rate of depurination under the present conditions is not known, but extrapolation from conditions for the monitored depurination of isolated ~X174 DNA (70°C, pH 5.0) predicts the production of 1 apurinic site per every 40 min. This extrapolation rests on several uncertainties. The value used for the activation energy 31 +_2 kcal/mole, was that determined for double-stranded DNA, and it is not known how charge-distributions and the availability of water within the virus capsid will affect the depurination rate. Therefore, the observed inactivation rate is in reasonable agreement with that
22 predicted on the basis o f depurination. In their study, Bleichrodt et al. (1968), reasoned that both phases o f the inactivation curve were due to protein damage. They concluded that only at lower temperatures ( < 6 0 °) a contribution of DNA damage did seem likely. Our results are not necessarily in contradiction, since our experiments were performed at low temperature (50°C) to limit protein damage. We furthermore used a low pH to increase depurination. The initial, more rapid inactivation seen in Fig. 1 does presumably reflect protein damage. In conclusion, heat treatment of bacteriophage ~X174 at acidic pH is highly mutagenic when assayed on a pre-irradiated host. It is likely that apurinic sites form a major contribution to this mutagenesis for the following reasons. The rate of inactivation can be closely predicted on the basis of known and extrapolated rates o f depurination, Secondly, a strong analogy is observed with the similarly SOSdependent transfection system. In this system, the involvement of apurinic sites could be convincingly shown. The mutagenic response with intact bacteria, if expressed per inactivation event, is about 5-10-fold higher than in the analogous transfection system with spheroplasts. This may suggest that spheroplasts apparently possess a limited ability to effectively express the induced functions. At the same time, this shows even more dramatically than before, the potential significance o f apurinic sites for heat mutagenesis or mutagenesis in general. This study was supported by grants from the National Institutes of Health (CA 24845, CA24998) and the National Science Foundation (PCM7680439).
References Baltz, R.H., P.M. Bingham and J.W. Drake (1976) Heat mutagenesis in bacteriophage T4: The transition pathway, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1269-1273. Bingham, P.M., R.H. Baltz, L.S. Ripleyand J.W. Drake (1976) Heat mutagenesis in bacteriophage T4: The transversion pathway, Proc. Natl. Acad. Sci. (U.S.A.), 73, 4159-4163. Bleichrodt, J.F., and E.R. and Abkoude (1967)IThe transition between two forms of bacteriophage ¢bX174 differing in heat sensitivity and adsorption characteristics, Virology, 32, 93-102. Bleichrodt, J.F., J. Blok and E.R. Berends-van Abkoude (1968) Thermal inactivation of bacteriophage ¢bX174 and two of its mutants, Virology, 36, 343-355. Kunkel, T.A., C.W. Shearman and L.A. Loeb (1981) Mutagenesis in vitro by depurination of ~X174 DNA, Nature (London), 291, 349-351. Lindahl, T., and B. Nyberg (1972) Rate of depurination of native deoxyribonucleicacid, Biochemistry, 11, 3610-3617. Radman, M. (1974) Phenomenologyof an inducible mutagenic DNA repair pathway in Escherichia coil: SOS repair hypothesis, in: L. Prakash, F. Sherman, M.W. Miller, C.M. Lawrence and H.W. Taber (Eds.), Molecular and Environmental Aspects of Mutagenesis, Thomas, Springfield, IL, pp. 128-142. Schaaper, R.M., and L.A. Loeb (1981) Depurination causes mutations in SOS-induced cells, Proc. Natl. Acad. Sci. (U.S.A.), 78, 1773-1777. Shearman, C.W., and L.A. Loeb (1979) Effects of depurination on the fidelityof DNA synthesis, J. Mol. Biol., 128, 197-218. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible repair in Escherichia coli, Bacteriol. Rev., 40, 896-907.