In this week BMC Seminar Antón Ameneiro-Álvarez PhD student, supervised by Stefán Þ. Sigurðsson, will give a presentation about RNA polymerase II transcription elongation and DNA damage (Icelandic title: Umritun með RNA pólýmerasa II og DNA viðgerð).

Abstract:  The genetic material of all living cells is constantly subjected to damage of both endogenous and exogenous origin, which can cause RNA polymerase to pause or stall. In vitro studies have shown that RNAPII is prone to pause or stall during the elongation phase of transcription. Such pausing during transcription elongation will result in retrograde motion of the protein complex on the DNA template. If the RNAPII moves into this backtracked state, the active site of RNAPII will lose contact with the 3´ end of the RNA. For continued transcription the elongation complex has to realign its active site with the nascent 3´ RNA end. However, if the pausing in the backtracked state is prolonged the complex can irreversibly stall and additional help for reactivation is required.

Various obstacles including nucleosomes, intrinsic secondary structures in the DNA and DNA damage can cause the polymerase to pause or stall. To overcome these challenges cells have developed a range of DNA repair pathways to deal with different kinds of DNA damage. The nucleotide excision repair (NER) system is conserved from yeast to humans and removes bulky adducts from DNA. Interestingly, NER occurs more rapidly in active genes and in the transcribed strand of these genes.

This sub-pathway of NER is named transcription-coupled nucleotide excision DNA repair (TC-NER) and depends on actively transcribing polymerase and specific TC-NER factors: Mfd in E.coli, and the DNA dependent ATPases of the Swif2/Snf2 family Rad26 (yeast) and CSB (human). Human cells containing mutations in the Cockayne syndrome B (CSB) gene show a defect in TC-NER but not in global genome repair. Patients with mutations in CSB present increased photosensitivity, impaired physical and sexual development and mental retardation.

The DNA dependent ATPase activity of Rad26/CSB and the fact that purified CSB binds to RNAPII raises the possibility that these proteins may promote passage of RNAPII through a variety of transcriptional impediments as Mfd does in E. coli.

However, little is known about the molecular mechanism of TC-NER or the biochemical activity of CSB/Rad26. Our results indicate that Rad26 is a DNA dependent translocase that is able to discriminate between different DNA structures showing a preference towards fork-like DNA structures, which stimulate Rad26’s ATPase activity.