Molecular Cell
Volume 24, Issue 2, 20 October 2006, Pages 245-255
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Article
A Mechanism of Nucleotide Misincorporation during Transcription due to Template-Strand Misalignment

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Summary

Transcription errors by T7 RNA polymerase (RNAP) may occur as the result of a mechanism in which the template base two positions downstream of the 3′ end of the RNA (the TSn+1 base) is utilized during two consecutive nucleotide-addition cycles. In the first cycle, misalignment of the template strand leads to incorporation of a nucleotide that is complementary to the TSn+1 base. In the second cycle, the template is realigned and the mismatched primer is efficiently extended, resulting in a substitution error. Proper organization of the transcription bubble is required for maintaining the correct register of the DNA template, as the presence of a complementary nontemplate strand opposite the TSn+1 base suppresses template misalignment. Our findings for T7 RNAP are in contrast to related DNA polymerases of the Pol I type, which fail to extend mismatches efficiently and generate predominately deletion errors as a result of template-strand misalignment.

DNA

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Present address: Laboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021.