282 



CHAPTF.R 21 



end and the removal oi a oucleotide at the 

 nucleotide end. i The diagram at the left of 

 the figure shows the </('P adding on to the 

 nucleoside end by the formation o\ a 3' 

 linkage between P* and the sugar at the end 

 of the chain. P-P being split oil the </CP*PP. 

 The diagram at the right shows </CP*PP 

 adding on to the nucleotide end o\ the chain 

 b\ linkage to the 5' position of the end nu- 

 cleotide which supplies the pyrophosphate 

 that splits off. In brief, the DNA strand 

 might be lengthened by the addition of a 

 nucleotide at either the 3' position of the 

 nucleoside end or at the 5' position of the 

 nucleotide end. 



It is possible to distinguish between these 

 two alternatives by treating the product of 

 a limited reaction first with DNase from 

 micrococci to enhance the action of another 

 added enzyme, splenic phosphodiesterase. 

 The latter enzyme degrades DNA by break- 

 ing the strand at all the 5' positions, so that 

 deoxyriboside 3'-monophosphates are pro- 

 duced. This position of breakage is indi- 

 cated by the arrows in Figure 21-2. If the 

 strand lengthens according to the diagram at 

 the right of the figure, radioactive P 32 should 

 be found in phosphate attached to deoxycy- 

 tidine. and P* should not be part of the 3'- 

 deoxyribotides of A. T. or G. If. on the 

 other hand, attachment is to the 3' position 

 at the nucleoside end of the strand, then, as 

 can be seen in the diagram at the left of the 

 figure, P* should not occur in inorganic 

 phosphates but should sometimes appear in 

 other deoxyriboside 3'-monophosphates be- 

 sides those containing C. The experiment 

 gives the latter result; that is, not only is 

 P* frequently present in all four kinds of 

 deoxyriboside 3'-monophosphates. but it is 

 absent from inorganic phosphate. 



An additional test of whether the DNA 

 strand grows at its 3' position involves treat- 

 ing the limited product with a different en- 

 zyme, snake venom diesterase. This en- 

 zyme digests DNA by breaking the bond 



^DNA 



P-P--P 



FIGURE 21-3. Degradation of a primer DNA 

 strand, which has grown at its nucleoside end, 

 by snake venom diesterase. The 3' positions 

 of degradation (arrows) occur sequentially 

 starting at the nucleoside end. 



between the phosphate and sugar at the 3' 

 position, starting at the nucleoside end of 

 the strand and proceeding toward the nucleo- 

 tide end. Thus, the DNA is gradually di- 

 gested into deoxyriboside 5'-phosphates, as 

 indicated by the arrows in Figure 21-3. 

 When the limited product is treated this way, 

 the result — as expected — is that almost all 

 the radioactivity has been removed from the 

 strand even though only a minute portion 

 of the DNA has been digested. Other re- 

 sults clearly show that the product of a lim- 

 ited reaction is DNA with one or very few 

 deoxyribotides added to the nucleoside end 

 of the strand. Still other evidence supports 

 the view that the 3' point of lengthwise link- 



