Figure 5.

Effect of O⁶-meG modified-DNA on transcription by hRNAPII. (A) Single and multiple rounds of transcription using HeLa nuclear extracts. Undamaged templates generated full-length (FL) transcripts of approximately 383 bases in length (lanes 1 and 2) while O⁶-meG-damaged templates produced two RNA species of 383 and 316 bases in length (lanes 3 and 4). A 50 bp DNA step ladder is shown (M). (B) Time course of transcription with hRNAPII past the undamaged DNA (lanes 1 through 8) and the O⁶-meG template (lanes 10 through 17). Aliquots were removed at the time points indicated on the figure to measure the extent of lesion blockage or bypass over time. Fragments were compared to a DNA standard of 175 bases in length used to minimize loading errors. Transcription past the damaged templates shows that the lesion partially obstructs RNA synthesis (only producing ∼60% of full-length RNA at time points 20 min through 60 min). The graph illustrates the % of full-length and truncated RNA over time (x-axis). It indicates that the amount of full-length and truncated RNA remain almost constant after the first ten minutes of the transcription reaction.