ABSTRACT
Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides 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 deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-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 for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.
Subject(s)
Bacteriophage T7/enzymology , DNA Primase/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Nucleosides/metabolism , Base Pairing/genetics , Base Sequence , Binding Sites , Catalysis , Chromatography, High Pressure Liquid , Conserved Sequence/genetics , DNA, Single-Stranded/chemical synthesis , DNA, Single-Stranded/chemistry , Deoxyribonucleosides/chemical synthesis , Deoxyribonucleosides/chemistry , Deoxyribonucleosides/genetics , Deoxyribonucleosides/metabolism , Genetic Engineering , Hydrogen Bonding , Inosine/analogs & derivatives , Inosine/chemistry , Inosine/genetics , Inosine/metabolism , Kinetics , Nucleosides/chemical synthesis , Nucleosides/chemistry , Nucleosides/genetics , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Oligoribonucleotides/biosynthesis , Oligoribonucleotides/genetics , RNA/biosynthesis , RNA/genetics , Substrate Specificity , Templates, GeneticABSTRACT
Abstract A new model to replace the Ogstron and tube reptation models for gel retardation of DNA is proposed that explicitly takes into account screening of the hydrodynamic interactions and polyelectrolyte effects. At short DNA sequence lengths, significant anomalous migration is predicted whose onset is dependent on the size of polyacrylamide gel pores. Thus, a 2-residue fragment has the same electrophoretic mobility as a 12-residue fragment for a polyacrylamide gel with a mesh size of 60Å. The oligonucleotide length at which anomalous migration is observed also depends on pore size. Experimental measurement of gel mobility for DNA fragments of the form N(pN)(n), where n = 1-11, 14 and 19 substantiate this phenomenon.
