1. Evidence Consistent with Similar Rates for Ts and Td
ADDENDUM: SUPPORTING INFORMATION
1. Evidence Consistent with Similar Rates for ts and td
Although A-T sites in T4 phage do not express ts, human A-T sequences exhibit a high propensity for expressing deletions (Shibata et al., 1994; Riccio et al., 1999).Additionally, populations of T4 phage exhibiting ts at *G-*C and G'-C' sites at 20C are also accumulating time-dependent lethals at the relatively high rate of 3.2103 lethal events per 24 hr (Drake, 1966). Deletion of superposition *A-T* sites (Fig. 2) in coding regions of T4 DNA would create frame shifts, which would be generally lethal and thus not detectable as viable mutations in these studies of T4 phage mutagenesis (Drake, 1966; Drake and McGuire, 1967; Bingham et al., 1976; Baltz et al., 1976; Drake and Baltz, 1976). Since about 66% of the 1.8105 bp genome of T4 phage is A-T (i.e., 1.188105 A-T pairs; Drake, 1966), one can estimate a rate, K(20o)AT, for populating A-T sites with superposition *A-*T states, using the relation
K (20o)AT = (3.210-3 events per 24 hr)/(1.188105 A-T pairs per genome) = 2.710-8 lethals/A-T/24 hr,
where each superposition *A-*T pair is assumed to be expressed as a lethal deletion. Since substitution lesions at *G-*C and G'-C' sites, measured as rIIr+ and r+r, accumulate about 20-fold faster at 20o than at 0oC (Drake, 1966), one could anticipate a proportional linear increase in the K(20o)AT value, for time-dependent lethal events at 37oC. A 17-fold increase yields a K(37o)AT estimate as
K(37o)AT = 4.6107 lethals/A-T/24 hr,
which is in order-of-magnitude agreement with the estimate for populating *G-*C and G'-C' sites with superposition enol-imine states at 37oC, i.e., K (37o)GC = 8.4310-7ts /GC/24 hr (Cooper, 2009). If superposition *A-*T states were processed like superposition *G-*C states, one could expect the quantum duplex of *A20# -*T0222 (Fig. 2) to similarly yield transcription and/or replication products corresponding to normal C00222 as suggested in Table 1. This, however, is not observed, implying discrimination against superposition *A-*T states by transcriptase quantum processing. These data (Drake, 1966) demonstrate that populations of T4 phage exhibiting ts at *G-*C and G'-C' sites are also accumulating time-dependent lethal events at rates consistent with molecular clock deletions, td, at superposition *A-*T sites.
2. Reequilibration Repair, *C2022→ C00022, of Time-Altered *C in the Second Round of Growth
After rD19 mutants were subjected to a metabolically inert incubation (114 hr at 31oC) and plated directly on a nonpermissive host, the yield was 69 revertants (Table 2), all of which originated as consequences of quantum state cytosine, *C2022, with thyminelike charge configurations on T-strands, retaining their thymine-analog properties for the initial transcription and subsequent replication of the decohered isomer, *C2022. If these decohered thyminelike cytosines, *C2022, retain their uniqueness, enabling them to function as thymine analogs, they will form complementary mispairs with normal adenine, *C2022–A002#, in the second round of replication in permissive hosts. Consequently, their contribution to the population of passaged rD19 revertants will be half the number of unpassaged revertants, which for rD19 is 34.5 (69/2) (Table 2). If a fraction of the initial 69 revertant lesions were not stable in the second round of replication, the resulting contribution to the passaged revertant population would be less than the expected 34.5. The revertant yield of incubated and passaged rD19 mutants shown in Table 2 is 28, or 6.5 fewer revertants (per 107 survivors) than expected, which is consistent with repair of time-dependent mutation in the second round of replication. Also after incubation periods (744 hr. at 45oC and 40 hr. at 58oC) at pH 7.25 and direct plating on nonpermissive hosts, the two rUV7 systems (Table 2) yielded 2700 and 1100 revertants, all of which are consequences of molecular clock lesions on T-strands. Contributions of stable revertant lesions on T-strands should be 1350 and 550, for the two rUV7 populations in Table 2. Actual yields of 1200 and 430 indicate that 150 and 120 fewer revertants, respectively, were produced than expected.
Mutant systems such as rD19 and rUV7 (Table 2) in which revertant yields after heat intervals and passage are less than half the yields from incubated but unpassaged phage require a modified equation for expressing the total yield of postpassaged revertants in terms of a sum of contributions of revertant lesions from T-strands and C-strands. To account for reduced yields of revertants from T-strand contributions, this equation can be written as
Observed (postpassage) = C-strand + T-strand (predictrepair)
where T-strand (predict-repair) is the number of revertants predicted from T-strand contributions as a consequence of passage (half the revertant yield from heated but unpassaged) minus the number of revertant lesions on T-strands that were repaired during the second round of replication on nonpermissive hosts. This allows estimates of the relative percentages of revertant point lesions on T-strands that were repaired after an initial growth on a nonpermissive host. By expressing revertant data of rD19 and the two rUV7 systems of Table 2 in these terms, one can estimate repair of revertant point lesions due to reequilibration of timealtered cytosines, *C2022, on T-strands (Table 3).
These data are consistent with about 19% repair of molecular clock events on T–strands in rD19. Similarly, rUV7 revertants from a pH 7.25 environment show an 11% repair after 744 hr. at 45oC and a 22% repair of such lesions after 40 hr. at 58oC. However, after pH 4.6 incubation of rUV7 for 24.5 hr. at 31oC (Table 3), the 180 postpassaged revenants originated from the sum of 165 lesions on T–strands plus 15 such lesions on C-strands where T-strand lesions did not exhibit repair. After an evolutionarily generated superposition of enolimine *G-*C states is decohered into an ensemble of *G and *C classical isomers, replication proceeds according to Topal and Fresco (1976) where complementary mispairs are formed. Consequently, enol and imine tautomers introduced as molecular clock substitutions would be expected to exhibit reequilibration repair in subsequent rounds of replication, consistent with observation. However, these data are inconsistent with the deamination of cytosine (5HMC) explanation of time-altered *C (Baltz et al., 1976; Drake and Baltz, 1976) since deaminated cytosine would not exhibit repair as observed (Table 3).
An absence of *C repair in rUV7 at pH 4.6 can be attributed to the increased H+ ion concentration. This would cause the electron lone-pair on the ring nitrogen in *C (Fig. 3) to be continually occupied by an available proton. Consequently, the imine double bond would be prevented from reassociating into the ring, thereby preventing reprotonation of the imine side chain. However, rD19 revertants exhibit reequilibration repair of time-altered *C at pH 4.6.
Addendum References
Riccio, A., Aaltonen, L., Godwin, A., Loukola, A., Percesepe, A., Salovaara, R., Masciullo, V., Genuardi, M., Paravatou-Petsotas, M., Bassi, D., Ruggeri, B., Andres, J., Klein-Szanto, P., Testa, J., Neri, G., Bellacosa, A. 1999. The DNA repair gene MBD4 (MED1) is mutated in human carcinomas with microsatellite instability. Nature Genet. 23:266-268.
Shibata, D., Peinado, M.A., Ioniv, Y., Malkhosyan, S., Perucho, M. 1994. Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation. Nature Genet. 6:273-281.