EFFECTS OF BASE ANALOG SUBSTITUTIONS IN THE NONCODING DC OF THE 3'-D(CTG)-5' TEMPLATE RECOGNITION SITE OF THE BACTERIOPHAGE-T7 PRIMASE

The 63-kDa gene 4 protein (DNA primase) of bacteriophage T7 catalyzes the synthesis of the oligoribonucleotides pppACC(C/A.) and pppACAC at single stranded DNA recognition sites 3'-d[CTGG(G/T)]-5' and 3'-d(CTGT G)-5', respectively. At these sites, the 5'-terminal deoxycytidine res idue is conserved but noncoding; the 3'-dC residue is required to init iate catalytic synthesis of oligoribonucleotides, yet it is not used a s a template residue for the synthesis of a complementary G residue in the RNA primer. We have examined the interactions between T7 primase and the functional groups of the 3'-dC residue by measuring the abilit y of the primase to catalyze the synthesis of oligoribonucleotides on synthetic single-stranded 20-mer templates [e.g., 3'-d(GCTATGGTGACTGGT AGTCG)-5'] that contain analogs of dC in the conserved pentanucleotide recognition site. Recognition sites containing 5-methyldeoxycytidine (m(5)dC) or 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (d(H4)C) substit utions for dC support oligoribonucleotide synthesis whereas those cont aining deoxythymidine (dT) and deoxyuridine (dU) substitutions do not. Oligoribonucleotide synthesis on the native template (containing dC) is inhibited competitively by the template containing a dT residue in the primase recognition site, 3'-[(N-10)TTGGT(N-5)]-5', with an appare nt K-i of 1.30 +/- 0.04 mu M. Templates containing dU residues, 3'-[(N -10)UTGGT(N-5)]-5' and 3'-[(N-9)UTTGGT(Ns)]-5', affect both the appare nt K-m and V-max parameters for oligoribonucleotide synthesis on the 3 '[(N-10)CTGGT(N-5)]-5' template.