Abstract
The transcription repair coupling factor (TRCF) of Escherichia coli has the so-called helicase motifs, is a DNA-, RNA Pol-, and UvrA-binding protein, and is required for the coupling of repair to transcription. We investigated the potential helicase, transcription termination, and transcription-repair coupling activities of TRCF on various substrates. We found that TRCF does not have a helicase activity on any of the substrates tested. However, the TRCF releases both RNA Pol and the truncated transcript from a transcriptional road block caused by a lesion, a "missing base," or a DNA-bound protein. It does not have any effect on rho-dependent or rho-independent transcriptional termination. However, some premature terminations were induced by TRCF at other sites. The coupling of transcription to repair occurs with supercoiled and relaxed circular DNA and with linear DNA. However, the coupling with linear DNA is strongly affected by the length of the DNA and does not occur with fragments in which the lesion is closer than 90 nucleotides to the 5' terminus of the template strand. Under transcription conditions the repair of lesions in the promoter region and up to the eleventh transcribed base is inhibited even in the presence of TRCF. Stimulation of repair in the transcribed strand starts at lesions at +15 nucleotides. Stimulation of repair occurs via facilitating the delivery of the A2B1 complex to the lesion site by the TCRF and can be inhibited by excess UvrA which binds to the TRCF off DNA. In vitro, strand-specific repair is not dependent on the MutL and MutS proteins which have recently been implicated in preferential repair in vivo.
Highlights
From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
The coupling with linear DNA is strongly affected by the length of the DNA and does not occur with fragments in which the lesion is closer than 90 nucleotides to the 5' terminus of the template strand
We investigated the effects of transcription repair coupling factor (TRCF) for helicase activity as well as for activities relating to rho factor such as releasing RNA Pol from pause sites, from protein blocks, or from ternary complexes formed as a result of a "missing base."
Summary
The transcription repair coupling factor (TRCF) of Escherichia coli has the so-called helicase motifs, is a DNA-, RNA Polo, and UvrA-binding protein, and is required for the coupling of repair to transcription. The TRCF releases both RNA Pol and the truncated transcript from a transcriptional road block caused by a lesion, a "missing base," or a DNA-bound protein. It does not have any effect on rhodependent or rho-independent transcriptional termination. A transcription-repair coupling factor (TRCF)l encoded by the mfd gene (Selby et at., 1991) releases the stalled RNA polymerase and promotes the preferential repair of the transcribed strand (Mellon and Hanawalt, 1989; Selby and Sancar, 1991, 1993).
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