Replication of DNA in Vitro 



325 



the same type of increase in ultraviolet 

 absorption following digestion with pancre- 

 atic DNAase. 



If the in vitro synthesis occurs in the same 

 way as it does in vivo, one might expect that 

 single-stranded DNA would serve as a better 

 primer than does double-stranded DNA. 

 Thus, we might expect the single-stranded 

 DNA, isolated from the virus </)X174, to be 

 capable of acting as primer. In fact, such 

 material is excellent primer, and heat- 

 treated DNA is better primer than unheated 

 DNA. Moreover, the preparations contain- 

 ing the most active ''synthesizing" enzyme, 

 which can be called a polymerase, will not 

 work with double-stranded primer DNA un- 

 less this is first heated or treated with DNAase. 

 Finally, the products of such syntheses be- 

 have as though they are primarily two- 

 stranded. 



In view of these results we may conclude 

 that the physical characteristics of the DNA 

 synthesized in vitro and in vivo are indistin- 

 guishable. Synthesis clearly involves single 

 chains which produce double chains probably 

 held together by H bonds. 



We can also study the detailed chemical and 

 physico-chemical characteristics of the in 

 vitro synthesis of DNA. If single chains pro- 

 duce double chains by forming complemen- 

 tary structures, then the capacity to form a 

 complementary chain should depend upon 

 the presence of purine and pyrimidine bases 

 in the substrate which can form appropriate 

 H bonds with the bases in the primer. Fig- 

 ure 35-5 shows some pyrimidine and purine 

 bases that do not naturally occur in DNA as 

 well as the four principal types that do. The 

 unnatural bases include uracil and 5-bromo 

 uracil (both of which might be expected to 

 have the same H-bonding capacities as thy- 

 mine), 5-methyl cytosine and 5-bromo cyto- 

 sine (both of which might be expected to have 

 the same H-bonding capacities as cytosine), 

 and hypoxanthine (which has two of the 

 three sites for H-bonding found in guanine). 



If an A in the single-strand primer dictates 

 its complement, by specifying that the comple- 

 mentary base provide the proper sites for H- 

 bonding A, then we would expect that uracil, 

 or 5-bromo uracil, could be substituted for 

 the thymine in thymidine 5'-triphosphate. 



When substrate containing deoxyuridine 

 5'-triphosphate (or 5-bromo deoxyuridine 

 5'-triphosphate), JCPPP, ^APPP, and c/GPPP 

 were employed, DNA synthesis was sup- 

 ported. Similarly, 5-methyl cytosine and 5- 

 bromo cytosine could substitute for cytosine. 

 On the other hand, the substitution of hypo- 

 xanthine for guanine did not support DNA 

 synthesis as well as did the other substitutions 

 mentioned. This would be expected, on the 

 hypothesis under test, since the former has 

 one fewer site for H-bonding than the latter. 

 Moreover, as expected, neither uracil nor 

 5-bromo uracil would substitute for C, A, or 

 G in (/CPPP, c^APPP, or ^GPPP, respectively. 

 Both 5-methyl and 5-bromo cytosine replace 

 cytosine specifically. Finally, hypoxanthine 

 replaces only guanine. Although hypoxan- 

 thine has the same H-bonding groups as thy- 

 mine, it does not replace thymine, probably 

 because the A-hypoxanthine pair, being com- 

 posed of two purines, takes up too much 

 space to fit the regular double helix configura- 

 tion. These results support the hypothesis 

 that normally the in vitro synthesis of DNA 

 is dependent upon the formation of comple- 

 mentary purine-pyrimidine pairs — A with T, 

 and C with G — just as is the case in vivo. 



It is also possible to analyze chemically 

 the DNA synthesized in vitro from the usual 

 four deoxyriboside 5 '-triphosphates. The 

 analysis shows that in the in vitro synthesized 

 DNA, A = T and C = G, just as they do 

 in natural DNA, even though the relative 

 concentration of the four triphosphates in 

 the substrate was distorted widely. Not only 

 do total pyrimidines = total purines in 

 "synthetic" DNA, as described, whether a 

 moderate or a large amount is synthesized, 

 but the particular A + T/G + C ratio found 



