of Work: The objective of this project is to examine whether transcription by RNA polymerase II may contribute to the high variation observed in the HIV genome and to investigate if transcriptional infidelity may affect the stability of genetic information in the cell. We have developed an in vitro system that allows quantitative evaluation of the fidelity of RNA polymerases. We have employed this system to examine the role of the non-transcribed strand on the fidelity of transcription using as a model system T7 RNA pol. The results indicate that unpaired bases in the non-template strand affect the frequency of frameshift errors during transcription. Transcription of DNA containing a 4-nucleotide loop in the non-transcribed strand resulted in a 16-fold increase in the frequency of 2-base additions relative to the frequency of additions on homoduplex DNA. We extended these observations to transcription with DNA substrates containing unpaired nucleotides in the template strand or a gap in the non-template strand. The results suggest that interactions of the RNA polymerase with the non-transcribed strand affect the fidelity of transcription. This is the first result suggesting that interactions between the RNA polymerase and the non-transcribed strand may affect transcription fidelity. This raises the question of whether bulky damage on the non-transcribed strand, which is not subject to transcription-coupled repair, may modify the fidelity of transcription. Experiments have been designed to examine this possibility.
