Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Transcription Blockage by Single-strand Breaks in Various Sequences and the General Model for Transcription Blockage by R-loop Formation
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 non-template 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 non-template 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 non-template 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 non-template strand break, the resulting blockage is greately enchanced (Belotserkovskii et al., 2013). Based upon these and other observations, we suggest that transcription blockage by breaks in the non-template 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.
This research was supported by NIH grants; CA077712 from the National Cancer Institute to P.C.H., and GM60987 from the National Institute of General Medical Sciences to S. M. M., and Undergraduate Research Grants at Stanford to A.J.N., S.S., and J.H.S.S.
Belotserkovskii, B. P., Neil, A. J., Saleh, S. S., Shin, J. H., Mirkin, S. M., & Hanawalt, P. C. (2013). Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks. Nucleic Acids Res, 41(3), 1817-1828.
Hanawalt, P. C., & Spivak, G. (2008). Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol, 9(12), 958-970.
Neil, A. J., Belotserkovskii, B. P., & Hanawalt, P. C. (2012). Transcription blockage by bulky end termini at single-strand breaks in the DNA template: differential effects of 5' and 3' adducts. Biochemistry, 51(44), 8964-8970.
Boris P. Belotserkovskii 1
1 Department of Biology