Transcription-coupled repair is an essential pathway for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA Polymerase II (Pol II). Mutations impairing CSB function cause severe genetic disease. Yet, the mechanism by which CSB powers Pol II bypass of certain lesions while triggering excision of others had remained unknown. Here we build structural models of Pol II bound to the CSB ortholog Rad26, including previously unmodeled regions, in nucleotide-free and bound states. This enables extensive molecular dynamics simulations and graph-theoretical analyses to define Pol II-Rad26 partitioning into dynamic communities and delineate how Rad26 structural elements function together to remodel DNA. We identify a key allosteric pathway coupling the motions of the Rad26 ATPase modules to conformational changes in Pol II and DNA, unveiling a structural mechanism for CSB-assisted Pol II progression past lesions. Our models allow functional interpretation of disease mutations associated with the genetic disorder Cockayne syndrome.

<b>Cryo-EM based Pol II–Rad26 structure model reveals the critical anchoring roles of the NTD and CTD domains. </b> <b>a</b>, Schematic showing the Rad26 protein domains along its sequence. Newly modeled regions are highlighted with solid black lines. Seven canonical super family 2 (SF2) helicase motifs are shown in red bars. <b>b</b>, View of Rad26 in cartoon representation colored by domains. <b>c</b>, Side view of the Rad26 active site cleft with DNA (blue) and highlighted functional motifs I–VI (red). <b>d</b>, Anterior and posterior views of Pol II–Rad26 complex. Rpb1, Rpb2 and Rpb4/7 are in light purple, brown and red, respectively. Circles demark zoomed-in regions in panels e-g. <b>e</b>, NTD of Rad26 interacting with the Rpb1, Rpb2 and Rpb4/7 subunits of Pol II. <b>f</b>, CTD and HD2-1 of Rad26 interacting with Rpb2 and DNA. <b>g</b>, NTD of Rad26 interacting with the Rpb1 protrusion and the clamp coiled-coiled domain.

Cryo-EM based Pol II–Rad26 structure model reveals the critical anchoring roles of the NTD and CTD domains. a, Schematic showing the Rad26 protein domains along its sequence. Newly modeled regions are highlighted with solid black lines. Seven canonical super family 2 (SF2) helicase motifs are shown in red bars. b, View of Rad26 in cartoon representation colored by domains. c, Side view of the Rad26 active site cleft with DNA (blue) and highlighted functional motifs I–VI (red). d, Anterior and posterior views of Pol II–Rad26 complex. Rpb1, Rpb2 and Rpb4/7 are in light purple, brown and red, respectively. Circles demark zoomed-in regions in panels e-g. e, NTD of Rad26 interacting with the Rpb1, Rpb2 and Rpb4/7 subunits of Pol II. f, CTD and HD2-1 of Rad26 interacting with Rpb2 and DNA. g, NTD of Rad26 interacting with the Rpb1 protrusion and the clamp coiled-coiled domain.

Chunli Yan

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