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J. Biol. Chem., Vol. 278, Issue 21, 19558-19564, May 23, 2003

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Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand^*

Virginia S. Kalogeraki  , Silvia Tornaletti  and Philip C. Hanawalt  ¶

From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.

Received for publication, January 31, 2003 , and in revised form, March 18, 2003.

^¶ To whom correspondence should be addressed. Tel.: 650-723-2424; Fax: 650-725-1848; E-mail: hanawalt{at}stanford.edu.

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