31. SYNTHESIS OF POLYNUCLEOTIDES 131 



absolute requirement for a primer, what is the meaning of the lag period? 

 One interpretation might be that the small amount of polynucleotide ma- 

 terial which is still present in the enzyme preparations 38 has to be trans- 

 formed to a suitable primer, perhaps by the action of a contaminating 

 nuclease. Further, what are the mechanisms of the synthetic reactions when 

 polynucleotides and oligonucleotides bearing 3'-phosphomonoester end 

 groups serve as primers? Can a polynucleotide chain be synthesized de novo 

 through the agency of this enzyme and can the primers which may not 

 actually be incorporated into the synthetic polymers influence the compo- 

 sition of the latter? 



O 



O y II /_ 



II /_ p— o 



P— o / 



o 



CH 2 /°\ R 



O 



I o 



o_l 



O OH 



H— O OH II _^||/V'H + 



0/ _ O— P— O P— o 



*-n||/_ ^ I I 



O— P— O OH O 



O O— CH 2 / U \ R' CH 2 / /U \ R 



OH OH OH OH 



Stepwise Reaction Catalyzed by Polynucleotide Phosphorylase 



The behavior of GDP in the enzyme system is unique. No polymeriza- 

 tion occurs when present alone even with the crude preparation which 

 readily polymerizes other nucleoside diphosphates. However, incorporation 

 in a polymer chain readily occurs when other nucleoside diphosphates are 

 also present . Interesting are the findings of Heppel and co-workers 41a ' 56 on the 

 polymerization of GDP using oligonucleotides such as pApApA as primer. 

 The reaction although slower than that with other nucleoside diphosphates 

 does lead to the stepwise addition of guanosine-o'-phosphoryl residues to 

 the expected end of the primer to give pApApApG and higher polymers. 



Another poorly understood reaction catalyzed by polynucleotide phos- 

 phorylase is the exchange of the terminal phosphate in nucleoside-5'-di- 

 phosphates with inorganic phosphate (P 32 -labeled) of the medium. 32 ' 33 ' 35 

 Does this reaction proceed via the true reversal of polynucleotide synthesis? 

 The specific case of GDP is again worth noting. With the Azotobacter en- 

 zyme, relatively crude 32 as well as highly purified, 56 exchange takes place 

 under appropriate conditions just as with other nucleoside diphosphates. 



