130 Transcription blockage by single-strand breaks in various sequences and the general model for transcription blockage by R-loop formation

Abstract

Transcription blockage can strongly affect gene expression and trigger other important biological phenomena like transcription-coupled repair (Hanawalt & Spivak, 2008). Thus, it is of interest to study the various factors that can cause transcription blockage and to elucidate mechanisms of their action. We studied T7 RNA polymerase (T7 RNAP) transcription blockage caused by single-stranded breaks localize either in the template or the nontemplate DNA strand (Belotserkovskii et al., 2013; Neil, Belotserkovskii, & Hanawalt, 2012). Partial T7 RNAP blockage was observed in both cases, but the patterns of blockage signals differed dramatically for these two types of lesions. A break in the template strand produces a sharp predominant blockage signal corresponding to the position of the break, as expected for an interruption in the DNA strand that is continuously tracked by RNAP during transcription. In contrast, a break in the nontemplate strand produces an irregular ladder of weak blockage signals that begins approximately at the position of the break and then extends far downstream from the break position, without either a predominant signal at the break position, or a pronounced downstream “end” of the ladder. The blockages produced by the break in the nontemplate strand increase dramatically when they are closely adjacent to G-rich homopurine sequences. These sequences cause partial transcription blockage, as we have previously established (Belotserkovskii et al., 2010); and in the presence of the nearby nontemplate strand break, the resulting blockage is greatly enhanced (Belotserkovskii et al., 2013). Based upon these and other observations, we suggest that transcription blockage by breaks in the nontemplate strand is due to their propensity to induce R-loop formation which destabilizes the transcription complex and renders it prone to spontaneous premature blockage/termination.