Transcription-coupled repair is an essential pathway for the removal of DNA lesions from the transcribed genome. The pathway is initiated by Cockayne Syndrome B protein (CSB) binding to stalled RNA polymerase II. Mutations impairing CSB function cause the severe genetic disease Cockayne syndrome. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase bypass of certain lesions while triggering excision of others is incompletely understood. Here we build cryo-EM based structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables extensive dynamics simulations and novel graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We support a mechanism wherein Rad26 binds to the upstream DNA duplex of a stalled Pol II, redirects the DNA path and recognizes the edge of the transcription bubble through conserved specific contacts. Nucleotide-driven closure and opening of the ATPase modules allow the Rad26 molecular motor to exert force on the upstream DNA duplex while alternating tight and loose DNA binding by the RecA2 domain causes pulling on the template strand. Analysis of &gt;30 ms of dynamics trajectories led to the discovery of a global allosteric network extending from Rad26 along the circumference of the Pol II central cavity. Thus, we support an allosteric mechanism for Rad26 to power distal conformational changes, which allow Pol II to bypass lesions in the transcribed strand. Our models allow functional interpretation of Cockayne syndrome disease mutations.

Molecular simulation sheds light on the mechanisms of transcription-coupled DNA repair

ACS Spring 2022

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