FURTHER STUDIES ON THE UNSTABLE AND “UNNATURAL” DESOXYRIBONUCLEIC ACIDS *

I n the fundamental work of I. K. Lehman and A. Kornberg (see also Bessman ~1 u1.I) on the enzymatic synthesis of desoxyribonucleic acid (DNA), two problems have a connection with the studies my associates and I have been conducting. One of these is related to the utilization of heated DNA as a primer in enzymatic synthesis. We have been subjecting to heat DNA that has transforming activity, and have found that the loss of biological activity always starts together with the loss of viscosity,2 even though different genetic markers in the same preparation have different heat ~tabilities.~ The important question of whether such hcating results in a loss of genetic information could not be answered definitely by these experiments, because a collapsed molecule might conceivably become biologically inactive in the transformation phenomenon and yet retain the information. Another problem is whether the information can change upon heating. We have presented evidence4 that one change that occurs in molecules heated in vitro is their unstabilization: the DNA remains biologically active, but loses its stability to heat. On reproduction, the injury is repaired and the molecules become stable again. Thus, the change was not a “mutation in nitro.” Our attempts to produce a true mutation in nitro (a change in DNA that is retained upon reproduction) have not yet led to positive results. The treatments used are heating, ultraviolet irradiation, and mild deamination with HNOz . Recently, however, we were able to demonstrate a very high mutability and gene unstabilization in vzvo with Escherichia ~ o l i , ~ and have advanced a working hypothesis that this is due to the unstabilization of DNA. Another aspect is the replacement of thymidylic acid by its analogues in the enzymatically synthesized DNA. The problem was to study the replacement of thymine in the DNA of living cells by the thymine analogues and to investigate the biological effects of such replacement.‘j-16 In the case of 5-bromouracil in a thymine-requiring strain, about one half of the thymine can be so replaced.‘j-* A substantial part of the thymine can also be replaced by 5-chlorouracil and 5-iodouracil (but not 2-thiothymine, 5-fluorouracil, or uracil). The amount of replacement, however, is smaller than in the case of 5-bromouracil; this amount is correlated with the similarity in size of the halogen substituent and the methyl group? The replacement of thymine by 5-bromouracil is highest in strains requiring thymine for g r ~ w t h ; ~ however, the replacement can be achieved in other cases of “disturbances” in DNA synthesis. Suchdisturbancesoccur naturally in several mutants (not only thyminerequirers, but also such forms as their “back-mutants”s and uracil-requirers)