Replication of DMA in Vitro 



323 



right), and a nucleoside end (bottom). (It is 

 possible to remove a nucleoside by a single 

 break at the 5' position at the nucleoside end 

 but requires the removal of a nucleotide at 

 the nucleotide end.) The diagram at the 

 left of the Figure shows the dCP* adding on 

 to the nucleoside end, by the formation of a 

 3' linkage between P* and the sugar at the 

 end of the chain, with P-P being split off the 

 i/CP*PP. The diagram at the right shows 

 JCP*PP being added to the nucleotide end of 

 the chain by linkage to the 5' position of the 

 end nucleotide which supplies the pyrophos- 

 phate that splits off. In brief, the DNA chain 

 might be lengthened by having a nucleotide 

 add on at either the 3' position of the nucleo- 

 side end or at the 5' position of the nucleotide 

 end. 



It is possible to distinguish between these 

 two alternatives in the following way. The 

 product of a limited reaction is treated first 

 with DNAase from micrococci in order to 

 enhance the action of another enzyme, spleen 

 phosphodiesterase, which is also added. The 

 latter enzyme degrades DNA by breaking the 

 chain at position 5', so that deoxyriboside 

 3'-monophosphates are produced. This posi- 

 tion of breakage is indicated by the arrows in 

 Figure 35-3. If the chain grows according 

 to the diagram at the right of the Figure, 

 radioactive P^- would be expected to be found 

 in phosphate attached to deoxycytidine. 

 Also, P* should not be found as part of the 

 3'-deoxyribotides of A, T, or G. If, on the 

 other hand, attachment is at the 3' position 

 at the nucleoside end of the chain, then, as 



>DNA 



>DNA 



FIGURE 35-3. Growth of a DNA chain at its nucleoside end (left) and nucleotide end 

 (right). Arrows show position of degradation by micrococcal DNAase plus splenic 

 phosphodiesterase. 



