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Fig. 5. Model depicting how transcription copes with irofulven lesions. Under normal conditions (upper-left panel), two main populations of Pol II coexist: the major one corresponds to a noncommitted fraction of Pol II, the second represents the fraction of engaged enzyme. Among the engaged fraction, two subtypes can be distinguished: the initiating Pol II, recognized by the H14 antibody, and the elongating subtype, recognized by the H5 antibody. The monoclonal H14 antibody specifically recognizes phosphorylated Ser5, whereas the H5 antibody specifically recognizes phosphorylated Ser2. Both serine residues are present within a heptapeptide that is repeated 52 times in the C-terminal domain of the human Pol II LS. In the presence of irofulven (upper-right panel), lesions are formed. These lesions are specifically processed by TCR after stalling of the RNA polymerase. Irofulven treatment is followed by an accumulation of initiating Pol II LS and a concomitant loss of the non-committed fraction of Pol II (lower-right panel). In contrast to the H14 species, the H5 fraction is rapidly lost. This suggests that the initiating Pol II cannot proceed efficiently to elongation. Three main hypothesis can be raised to explain this phenotype: (1) the presence of numerous lesions in the vicinity of the promoter region, (2) the inefficient recycling of other subunits that form, with Pol II LS, the holoenzyme and/or (3) the preferential association with repair sites of factors involved in both transcription and repair. Stalled polymerases become ubiquitylated and degraded through the proteasome. New protein synthesis is required to restore the initial pool of noncommitted enzyme necessary for transcription restart (lower-left panel). Proteasome inhibitors block Pol II LS recycling, inhibiting transcriptional recovery and increasing the probability of secondary DNA lesions. Cycloheximide (CHX), by blocking new protein synthesis, induces a rapid and complete loss of Pol II LS owing to the continuous degradation of newly engaged enzymes.
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