238 ERXST FREESE 



has Ihtii ()I)srrvc,l ( I^autz aii.l Fivcsc, 1960; Lett ct ni. 1962). The 

 g'dp might intc'i'tVic wilh 1)XA (luplication oi' cause tlic incorporation 

 of a wrong base. 



5. The depiii-inalcd DNA is labile at higli i)ll and as sliown in 

 Section II,C,3 for the (le|)ui'inati()n l)y low pli, may occasionally break 

 even at neutral i)II. Again this might induce larger alterations or be 

 lethal, but ]irobably does not induce jjoint mutations. 



Any one of these five different effects could be lethal. Treatment of 

 DNA with MM8 or DMS rapidly methylates the phosjihates and the 

 purine bases (Reiner and Zamenhof, 1957) and leads to the depoly- 

 merization of DNA (Lett et (iL, 19621. These agents are, however, 

 very slightly mutagenic (per lethal hit) for bacteriophages, in contrast 

 to the ethylating agents (Loveless, 19.59; Bautz and Freese, 1960). This 

 seems to exclude points 4 and 5 as the major cause of lethality, since 

 depurination by alkylation should be as mutagenic as by low pH 

 treatment. Of the remaining three mechanisms — 1, 2, and 3 — the hy- 

 drolysis of the DNA backbone (2) seems the most likely mechanism for 

 the major cause of lethality by methylation especially since Loveless 

 (1959) has shown that the phage titer still decreases after the treatment 

 with MMS has been stopped by sodium thiosulfate or by dilution. 



The inactivation by ethylating agents proceeds much more slowly 

 than for mcthylating agents, but for phages the frequency of induced 

 mutants per lethal hit is much larger for ethylating than for methylating 

 agents. Since the amount of esterification of the phosphates in DNA is 

 about the same as for methylation the high mutagenicity indicates that 

 DNA backbone breakage by mechanism 2 is much less for ethylated 

 than for methylated DNA. In agreement with this expectation Lett 

 et al. (1962) find that EMS treatment of DNA does not influence 

 its viscosity while MMS treatment leads to a rapid degradation. The 

 authors interpret these results differently; they believe that mechanism 

 5 is mainly responsible for breakage. EMS or EES (Benzer, 1961 ; E. B. 

 Freese, 1961) induce point mutations in phage T4. As for the nnitations 

 induced by low pH this finding makes it unlikely that they are caused 

 by breaks of the DNA backbone. Even in maize ethylating agents 

 mainly induce mutations of small genetic extent and not large altera- 

 tions (Heiner et al., 1960). 



For the induction of mutations mechanisms 1, 3, and 4 are then to be 

 considered. For mechanism 1 the alkyl group on the phosphate would 

 have to interfere in some way with the pairing properties of the bases 

 and cause the incorporation of a w^rong base during DNA duplication. 

 But the alkylated phosphate triester hydrolyzes and each removed ethyl 

 group should decrease the frequency of mutations in treated phages. 

 Since actually the contrary is observed (Bautz and Freese, 1960) mecha- 



