2SS 



( hap iik 21 



d-GC), is composed of two homopolymers; 

 one strand contains onlj C's and the other 

 onlj G's, the two strands base-pairing 

 to form a double helix. Whereas A = T 

 after extensive synthesis of dAT, most 

 products of dCklC synthesis show 56 to 

 81% dGP. 



Both dAT and dGdC illustrate that the 

 base sequence in certain strands is not ran- 

 dom. That such strands can have biological 

 significance is strikingly supported by the 

 discover} of a "natural dAT" polymer in 

 the sperm of a certain crab. Nearest- 

 neighbor analysis shows that this polymer, 

 which comprises about 30% of the total 

 DNA content, contains A and T in strict 

 alternation in 93% of the dinucleotide se- 

 quences. About 3% of the bases associated 

 with this natural dAT are G or C, and all six- 

 teen dinucleotide sequences are found. One 

 might suspect that the G- and C-containing 

 nucleotides are contaminants of the typical 

 DNA comprising 70% of the total. If they 

 are, extensive in vitro synthesis using natural 

 dAT as primer-template should occur just as 

 rapidly whether or not the substrate of 

 clAPPP and TPPP has </CPPP and </GPPP 

 added to it. One finds, however, that the 

 replication rate in the absence of the latter 

 two triphosphates is only 19% of that ob- 

 tained in their presence, supporting the view 

 that G and C bases are an integral part of 

 the "natural dAT" polymer. 



Calf Thymus DNA Polymerase 



All in vitro DNA syntheses so far discussed, 

 whether they occur de novo (by an un primed 

 initiation of single strands) or involve com- 

 plementary base-pairing (as in limited and 

 extensive syntheses ). require the presence of 

 E. coli DNA polymerase. The DNA poly- 

 merase isolated from calf thymus cannot 

 form dAT or dGdC de novo; in other words, 

 it is apparently incapable of unprimed DNA 



synthesis. 1 Some evidence has been ob- 

 tained that M-bond, base-pairing, template 

 synthesis by calf thymus DNA polymerase 

 begins once chain length exceeds twenty 

 monomers. The DNA polymerase from calf 

 thymus, like that from E. coli, uses DNA 

 as a template to make DNA which resembles 

 native DNA in primary and secondary struc- 

 ture, composition, sequence, and molecular 

 size; the synthesized DNA also does not 

 completely undergo strand separation upon 

 heat denaturation. 



Biological Replication in Vitro 



Insofar as extensive synthesis of DNA in 

 vitro is concerned, all, or almost all the 

 physical and chemical characteristics of the 

 product are consistent with the view that 

 this is a biological process. However, there 

 are several differences between the in vitro 

 and the in vivo processes. Apparently E. 

 coli DNA polymerase works more slowly 

 in vitro than in vivo. Calf thymus DNA 

 polymerase has a maximum yield of 100%. 

 Both enzymes make DNA which does not 

 strand separate completely when heat de- 

 natured; this behavior may be correlated with 

 the branched DNA made in vitro by the E. 

 coli enzyme. All these differences may re- 

 sult from contaminants in vitro which are 

 not present in vivo, and from changes which 

 occur in isolating the in vivo DNA for in 

 vitro syntheses. When these possibilities are 

 considered, there is little doubt that exten- 

 sive synthesis is performed in vitro in essen- 

 tially the same manner as in the living cell 

 and that it produces essentially the same 

 product. 



Finally, special attention should be given 

 to the DNA polymerases essential for the 

 extended biological synthesis of DNA. Pre- 

 viously-known enzymes are specific in that 

 they act upon one or a few particular sub- 

 strates usually modified in the same way. 



By N. Sueoka. 



•See F. J. Bollum (1963, 1964). 



