Figure 4. Effect of primer length on pyrophosphorolysis.

Pyrophosphorolysis by WT or mutant D₂₁₁N Ty1 RT were carried out for 60 minutes in the presence of either 10 mM Mg²⁺ or 2 mM Mn²⁺. Substrates are 5′ ³²P-end labeled primers of lengths 14, 20, 24, 27, 28-mer (RAG 998, RAG 1250, RAG 1249, RAG 1248, RAG 952 respectively, Fig. 1), each paired with the 28′-mer template oligo (RAG 1109, Fig. 1). A, shows WT reactions with different primer length substrates in the presence or absence of PPi. B, represents D₂₁₁N reactions with or without PPi. Control reactions on both the panels are in the absence of enzyme or Metal ²⁺. C and D represent bar graphs for WT and DN Ty1 RT reactions depicting ratios of intensities of cleaved products to the total products in reactions (I cleaved products/I total bands). Black bars are for reactions in Mg²⁺ while grey bars are for reactions in Mn²⁺.