The molecular architecture of human transcription-coupled DNA repair complexes

DNA repair is essential for the genomic integrity of cells and organism survival. DNA damage not only causes mutation but also can result in disease and lethality through obstruction of transcription by RNA polymerase II stalled at DNA lesions. Transcription-coupled repair (TCR) allows for rapid removal of DNA lesions that block transcription from actively transcribed regions of the genome. RNA polymerase II (RNAPII), transcription factor IIH (TFIIH), Xeroderma Pigmentosum complementation group-G protein (XPG), and Cockayne Syndrome complementation group-B protein (CSB) have been implicated as key players in the TCR pathway. The overall objective of this work is to understand how the TCR machinery assembles and functions. To this end we have begun to define the structural characteristics of these protein complexes through biochemistry and single particle electron microscopy analysis.; Human TCR proteins were purified and an in vitro transcription system was used to create artificially stalled RNAPII complexes. Electromobility shift assays (EMSA) demonstrated that stalled-RNAPII, XPG, and CSB formed a stable supra-molecular complex and sub-complexes. Further EMSA analysis with transcription reactions modified to lack the non-template DNA strand failed to form complexes between stalled RNAPII and the other TCR proteins. These findings in addition to results from our collaborators in the Cooper lab indicate that the DNA structure that RNAPII maintains during transcription is essential for TCR complex formation.; As a first step toward structural analysis of the supra-molecular TCR complex the human RNAPII enzyme was analyzed by electron microscopy. Single particle reconstruction methodologies confirm that the human RNAPII is similar, though not identical, to the yeast RNAPII in overall architecture. 2-D analysis further revealed that there is conformational heterogeneity in the sample.; These studies provide a platform on which we can build up higher order TCR complexes and obtain 3-D structures of these large macromolecular entities to obtain structural information and gain further insight into TCR.