Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3 '-(CTC)-5 ' and their effects on the activity of bacteriophage T7 primase

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotide s pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[ CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidin e residue is conserved but noncoding, A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and t he effects of these analogue templates on T7 primase activity have been exa mined. The nucleosides employed include a novel pyrimidine derivative, 2-am ino-5 -(beta-2-deoxy- D-erthylope-pentofuranosyl)pyridine (d2APy), whose sy nthesis is described. Template sites containing d2APy in place of the crypt ic dC support oligoribonucleotide synthesis whereas those containing 3-deaz a-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used f or a critical interaction by the enzyme. Recognition sites containing 4-ami no-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H,3H]-pyrimidion e (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribon ucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deo xyriboside (d(2H)T) substitutions do not, suggesting the importance of Wats on-Crick interactions at this template residue, Template sites containing 7 -deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG su pport oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibiti on studies suggest that the primase enzyme binds "null" substrates but cann ot initiate RNA synthesis.