Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 2. Errors in transcription by different polymerases resulting from ethenocytosine and its hydrated intermediate

Abstract

Chloroacetaldehyde-modified poly(rC) or poly(dC) was prepared containing either 8-36% 3,N4-ethenocytidine (.epsilon.C) or 8-36% of a mixture of .epsilon.C and the hydrated .epsilon.C (.epsilon.C.cntdot.H2O), with the hydrate greatly predominating (> 90%). These ribo- and deoxyribonucleotide templates were transcribed with DNA-dependent RNA polymerases from Escherichia coli and calf thymus, in the presence of either Mn2+ or Mg2+ and all 4 ribonucleoside triphosphates. All the polymers tested were transcribed with either cation present. In an earlier report, transcriptional ambiguities resulting from .epsilon.C residues in enzymatically synthesized poly(rC,.epsilon.rC) were studied with E. coli DNA-dependent RNA polymerase in the presence of Mn2+. The misincorporations were confirmed when poly(rC,.epsilon.rC) and poly(dC,.epsilon.dC), prepared by reaction of poly(rC) and poly(dC) with CAA, were transcribed in the presence of either Mn2+ or Mg2+. The presence of hydrated .epsilon.C in polymers also leads to misincorporations but with reproducible differences from those found with .epsilon.C alone. Nearest-neighbor analysis of the transcription products showed that the hydrate caused misincorporation of A > U .mchgt. C while .epsilon.C caused misincorporation of U > A .mchgt. C. The extent of misincorporation in transcription was less with Mg2+ than with Mn2+, but the pattern of ambiguity was the same with both cations and with both ribo- and deoxyribocytidylate polymers. Calf thymus DNA-dependent RNA polymerase IIB was also used to transcribe deoxyribocytidine polymers with Mn2+ as the cation. .epsilon.C and .epsilon.C.cntdot.H2O both caused a high level of misincorporation of U, A, and C, but the preferred misincorporations differed slightly from those found with E. coli DNA-dependent RNA polymerase. For both prokaryotic and eukaryotic enzymes, the type of misincorporation resulting from the loss of H bonding by modification of the N-3 of C not only differed between .epsilon.C and the hydrated intermediate but also both differed from the transcriptional errors resulting from the presence of 3-methylcytidine in poly(dC) or poly(rC). The errors made by these polymerases during transcription do not result primarily from the conditions used (cation, ribo- or deoxyribotemplate) but must be at least in part attributed to the enzyme recognizing some facet of the modified base other than the lack of normal H bonding.