Substitution of analogues in DNA synthesis. 



The second line of evidence is derived from studies of the activity of the 

 substrates when substitutions are made in the purine and pyrimidine bases. 

 From the many interesting reports on the incorporation of bromouracil (22), 

 azaguanine (23) and other analogues into bacterial and viral DNA, it might 

 be surmised that some latitude in the structure of the bases can be tolerated 

 provided there is no interference with their hydrogen bondings. When experi- 

 ments were carried out with deoxyuridine triphosphate or 5-bromodeoxy- 

 uridine triphosphate, it was found that they supported DNA synthesis when 

 used in place of thymidine triphosphate but not when substituted for the 

 triphosphates of deoxyadenosine, deoxyguanosine or deoxycytidine. As already 

 described (24), 5-methyl- and 5-bromocytosine specifically replaced cytosine; 

 hypoxanthine substituted only for guanine; and, as just mentioned, uracil and 

 5-bromouracil specifically replaced thymine. These findings are best interpreted 

 on the basis of hydrogen bonding of the adenine-thymine and guanine-cytosine 

 type. 



Along these lines it is relevant to mention the existence of a naturally oc- 

 curring "analogue" of cytosine, hydroxymethyl cytosine (HMC), which is 

 found in place of cytosine in the DNA of the coli bacteriophages of the T-even 

 series (25). In this case the DNA contains equivalent amounts of HMC and 

 guanine and, as usual, equivalent amounts of adenine and thymine. Of addi- 

 tional interest is the fact that the DNA's of T2, T4 and T6 contain glucose 

 linked to the hydroxymethyl groups of the HMC in characteristic ratios (26, 

 27, 28) although it is clear that in T2 and T6 some of the HMC groups contain 

 no glucose (27). These characteristics have posed two problems regarding the 

 synthesis of these DNA's which might appear to be incompatible with the 

 simple base-pairing hypothesis. First, what mechanism is there for preventing 

 the inclusion of cytosine in a cell which under normal conditions has deoxy- 

 cytidine triphosphate and incorporates it into its DNA? Secondly, how does 

 one conceive of the origin of the constant ratios of glucose to HMC in DNA 

 if the incorporation were to occur via glucosylated and non-glucosylated HMC 

 nucleotides? Our recent experiments have shown that the polymerase reaction 

 in the virus-infected cell is governed by the usual hydrogen-bonding restric- 

 tions but with the auxiliary action of several new enzymes developed specifi- 

 cally in response to infection with a given virus (29, 30). Among the new 

 enzymes is one which splits deoxycytidine triphosphate and thus removes it 

 from the sites of polymerase action (30). Another is a type of glucosylating 



s-59 



