Effects of DNA lesions on transcription elongation by T7 RNA polymerase

Abstract

T7 phage RNA polymerase was used to transcribe a series of DNA templates bearing any of several precisely localized lesions. Lesions were positioned downstream of the T7 promoter on either strand of the DNA template to investigate the effects of these lesions on elongation of transcription. The following four types of DNA modifications were studied: 1) 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), a synthetic apurinic/apyrimidinic site; 2) 8-oxoguanine (8-oxodG), an oxidized derivative of guanine; 3) N-acetyl-2-aminofluorene (AAF) modified guanine; 4) 2-aminofluorene (AF) modified guanine. None of these lesions blocked transcription elongation when they were located on the non-template strand. Lesions on the template strand blocked elongation with varied efficiency. The series of AAF-dG, AF-dG, and tetrahydrofuran lesions showed a progressively decreasing ability to block elongation, while 8-oxo-dG caused little, if any, premature termination. T7 RNA polymerase was able to read through all of the lesions with sufficient efficiency to permit chain termination sequencing using the read-through products as templates. AAF-dG and AF-dG adducts did not induce detectable misreading. Adenine and, more rarely, cytosine were incorporated opposite 8-oxo-dG, as observed for translesional synthesis by DNA polymerases. Adenine was most commonly inserted opposite the non-instructional abasic site analogue, although a minor fraction of guanine was incorporated.