NUCLEOTIDE-SEQUENCE CONTEXT EFFECT OF A CYCLOBUTANE PYRIMIDINE DIMER UPON RNA-POLYMERASE-II TRANSCRIPTION

We have studied the role of sequence context upon RNA polymerase II ar rest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cycl obutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II ) and initiation factors, We selected a model sequence containing a we ll characterized site for RNAP II arrest in vitro, the human histone H 3.3 gene arrest site, The 13-base pair core of the arrest sequence con tains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located a t the center of a dA(6).dT(6) tract eliminates bending (Wang, C.-I., a nd Taylor, J.-S. (1991) Proc. Natl. Acad Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arres t, We show that a CPD in the transcribed strand at either of two locat ions in the arrest site is a potent block to transcription, However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the exte nt of bending of the DNA and improve readthrough efficiency. These res ults demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.