222 ERNST FREESE 



mistakes in incoipoi'atioii (( l-C — * A-Ti inoic ficcniciitly than mistakes 

 in replication since in the lattci' ease BU is already a inemhei' of a DNA 

 strand and the electi'onetiativity of its bromine may become |)aitially 

 reduced by electron-donatino; nei^lil)or <2;i-oups. 



BUdR is a more etiicicnt mutaiicn than HV (Freese, 1959a; I.itmaii 

 and Pank^e, 1960) probably because it is more readily converted into 

 the deoxynucleotide triphosphate and interferc^s less with the formation 

 of ni'acii and cytosine. Both BU and Bl'dlv induce point nuitations 

 (Benzer and Freese, 1958; Frccse, 1959a). 



The jiroposed mechanism of nuitagenesis is supported by several 

 other obser\-ations. "NMien ])liap:e infected bacteria are exposed to BUdR 

 and diluted into a medium containint^ excess thymidine, befoi'e any 

 intact phages are formed, indueed mutations are still observed in the 

 lysate (Litman and I'aidee, 1960). The same is observed wlien jirotein 

 synthesis is stopped by chloramphenicol and BUdR is present only while 

 the phage DNA multiplies (Litman and Pardee, 1959; Brenner and 

 Smith, 1959). This shows that the induction of mutations by BUdR does 

 not reciuire some fixation process and does not depend on protein syn- 

 thesis. The observation of mottled plaques after BUdR mutation induc- 

 tion shows that only one base of a DNx\ pair is replaced at a time and 

 that DNA apparently duplicates by the separation of the complementary 

 strands (Pratt and Stent, 1959), as has been assumed. 



When DNA contains BU, its biological activity is much more sensi- 

 tive to chemical attack. This has been observed by its sensitivity to UV 

 (Litman and Pardee, 1960; Kozinski and Szybalski, 1959; Greer, 1960; 

 Lorkiewicz and Szybalski, 1960), to UV and visible light (Stahl, 1961), 

 and to X-rays (Djordjevic and Szybalski, 1960; Kaplan and Tomlin, 

 1960). This additional effect of BU incorporation is apparently only 

 lethal and not mutagenic. The same applies to the increased lethality 

 of BU-containing jihages wlien treated by hydroxylamine (Freese et nl., 

 1961b). 



2. 2-Aminopnrine (AP) 



AP is mutagenic, apparently by a quite different mechanism. It can 

 pair in its normal tautomeric form with two bases, with T by two hydro- 

 gen bonds and with C by one hydrogen bond (Freese, 1959a) (see Fig. 

 7). The pairing involving two hydrogen bonds should occur much more 

 frequently tlian that with one liydrogen bond since in the latter case 

 the two protonless nitrogens repel each other by their electron clouds 

 and their relative steric ])osition is not fixed, making it more difficult for 

 the polymerizing enzyme to link the nucleotide to the growing DNA 

 chain before it has s('parate(j again. Thus AP should l)e incorporated 



