Abstract
RNA polymerase II stalled at a lesion in the transcribed strand is thought to constitute a signal for transcription-coupled repair. Transcription factors that act on RNA polymerase in elongation mode potentially influence this mode of repair. Previously, it was shown that transcription elongation factors TFIIS and Cockayne's syndrome complementation group B protein did not disrupt the ternary complex of RNA polymerase II stalled at a thymine cyclobutane dimer, nor did they enable RNA polymerase II to bypass the dimer. Here we investigated the effect of the transcription factor 2 on RNA polymerase II and RNA polymerase I stalled at thymine dimers. Transcription factor 2 is known to release transcripts from RNA polymerase II early elongation complex generated by pulse-transcription. We found that factor 2 (which is also called release factor) disrupts the ternary complex of RNA polymerase II at a thymine dimer and surprisingly exerts the same effect on RNA polymerase I. These findings show that in mammalian cells a RNA polymerase I or RNA polymerase II transcript truncated by a lesion in the template strand may be discarded unless repair is accomplished rapidly by a mechanism that does not displace stalled RNA polymerases.
Highlights
Preferential repair is the repair of certain regions of the genome at a faster rate compared with the bulk of genomic DNA [1,2,3,4,5]
The purified CSB protein, in contrast to transcription-repair coupling factor (TRCF), does not disrupt the ternary complex of stalled RNA polymerase II but instead it appears to function as a transcription elongation factor for RNA polymerase II [10]
To test for the effect of HuF2 on RNA polymerase II, transcription was carried out with U-less cassette template, pMLU112 (Fig. 1), HuF2 was added to the reaction mixture, and the reaction was supplemented with UTP and incubated further
Summary
Preferential repair is the repair of certain regions of the genome at a faster rate compared with the bulk of genomic DNA [1,2,3,4,5]. In a study aimed at uncovering the mechanism of stimulation of repair by transcription, it was found that human RNA polymerase II stalled at a thymine dimer was rapidly dissociated from the template/ substrate by human cell-free extract without detectable stimulation of repair [11]. This observation raised the possibility that the basic mechanism of coupling repair to transcription in humans may be different from that of E. coli. Any model for transcription-coupled repair in humans must account for the presence of a relatively abundant nuclear factor that disrupts ternary complexes rapidly and efficiently
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