144 H. GOBIND KHORANA 



Enzymically and biologically the most important question resides in the 

 function of the primer DNA. The information gained in the limited syn- 

 thetic reaction shows that reaction occurs as above at the existing 3'- 

 hydroxyl end group of the primer chain, similar to the priming of poly- 

 nucleotide phosphorylase action by ribo-oligonucleotides. Although the 

 relationship between the limited and the extensive synthetic reactions is 

 not clear, it appears likely 92 that the same enzyme is involved in the two 

 reactions. Some support in favor of the idea that 3'-hydroxyl ends are re- 

 quired for the polymerase action may be derived from the observations 

 that already exist. Brief treatment of the primer DNA with pancreatic 

 deoxyribonuclease stimulates 86 about threefold the rate of the synthetic 

 reaction. Also highly pertinent are the observations of Bollum 105 with the 

 calf thymus polymerase (see below) that DNA preparations of rather low 

 viscosity from salmon sperm were effective as primers whereas "native" 

 DNA from calf thymus was not. 



The influence of primer DNA on the composition of the product and 

 the incorporation of purine and pyrimidine analogs point to the idea of 

 primer DNA serving as a template for the newly synthesized DNA. As 

 with the polynucleotide phosphorylase, the urgent question that needs to 

 be answered is whether new chains are synthesized by the polymerase sys- 

 tems. The finding of Bollum 105 (see also Lehman 106 ) that heating converted 

 non-priming DNA's to effective primers could be interpreted to mean that 

 a single-stranded DNA is in fact required as a primer and that the polymer- 

 ase catalyzes the synthesis of the complementary strand. The single- 

 stranded DNA of </>X174 phage 107 serves as primer and has been reported 

 not to require heat activation. 106 The function of unwinding double-stranded 

 DNA to a single-stranded DNA could then also be ascribed to pancreatic 

 deoxyribonuclease in the E. coli system. 



In the events that the primer is a single-stranded DNA and that the ac- 

 tion of the polymerase consists of the synthesis of the complementary chain, 

 then the synthetic reaction should cease when the amount of DNA has been 

 doubled. 105 DNA increases of more than twofold would be possible only if 

 the polymerase systems contained enzyme (s) capable of converting two- 

 stranded non-primer DNA to single-stranded primer DNA. 105 An alterna- 

 tive explanation for extensive synthesis would be that the synthetic reaction 

 involves, as mentioned above, additions to preexisting chains and that the 

 elongated chains are continually broken down by nucleases (phosphodies- 

 terases) with the liberation of new 3'-hydroxyl end groups. 



Bollum's recent experiments indicate that synthetic thymidine oligo- 



105 F. J. Bollum, J. Biol. Chem. 234, 2733 (1959). 



106 I. R. Lehman, Ann. N. Y. Acad. Set. 81, 745 (1959). 



107 R. L. Sinsheimer, /. Mol. Biol. 1, 43 (1959). 



