Abstract
Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.
Highlights
DNA template strand (t-strand) lesions that block elongation by RNA polymerase (RNAP), most notably UV-induced cyclobutane dimers (Bohr et al, 1985; Witkin, 1966), are targeted for preferential repair in a process called transcription-coupled repair [Transcription-coupled repair (TCR); (Hanawalt and Spivak, 2008; Mellon and Hanawalt, 1989; Mellon et al, 1987)]
In TCR, the stalled elongating RNAP serves as an efficient detector of t-strand lesions that becomes a privileged entry point into the nucleotide excision repair (NER) pathway through the action of a transcription repair coupling factor (TRCF)
Escherichia coli (Eco) Mfd was discovered through genetic analysis (Bockrath et al, 1987; Witkin, 1966), identified as a TRCF, purified, and biochemically characterized (Selby and Sancar, 1993; Selby and Sancar, 1994; Selby and Sancar, 1995a; Selby and Sancar, 1995b)
Summary
DNA template strand (t-strand) lesions that block elongation by RNA polymerase (RNAP), most notably UV-induced cyclobutane dimers (Bohr et al, 1985; Witkin, 1966), are targeted for preferential repair in a process called transcription-coupled repair [TCR; (Hanawalt and Spivak, 2008; Mellon and Hanawalt, 1989; Mellon et al, 1987)]. Escherichia coli (Eco) Mfd (mutation frequency decline – named for its activity to reduce the frequency of suppressor mutations that occur when protein synthesis is inhibited subsequent to mutagenic treatment) was discovered through genetic analysis (Bockrath et al, 1987; Witkin, 1966), identified as a TRCF, purified, and biochemically characterized (Selby and Sancar, 1993; Selby and Sancar, 1994; Selby and Sancar, 1995a; Selby and Sancar, 1995b) These experiments showed that Mfd was necessary and sufficient for TCR in vivo and in vitro and that it expressed two major activities: 1.
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