Transcrtiption of nicked DNA templates

Abstract

RNA polymerases can bypass certain nicks and gaps of DNA templates to certain extent during transcription. As a part of developing a novel DNA sequencing technology, we have quantitatively determined the efficiency of bypass and transcription by a single-subunit bacteriophage RNA polymerase over a nick of DNA templates located at various positions. Synthetic DNA template with an SP6 promoter were designed to contain one nick at a defined location on the lower template or upper coding strand of the DNA template. Hybridization of three synthetic oligomers, one of which was a full-length strand of 55 nucleotides, generated a template with a nick at a defined position. The entire length of the templates covers from the position -18 to +37, where transcription starts at +1 position. Nicks were designed to be located immediately downstream of -4, +1, +5, +9 and +19 on the upper strand, and +1, +6, +10, +14 and +24 on the lower strand. The hybridized templates with a nick at various positions were subject to transcription reactions by the bacteriophage SP6 RNA polymerase under the standard conditions. The effects of the nick on the transcription initiation and elongation efficiency were quantitatively measured as compared with the control DNA template without a nick. A nick located within the abortive initiation region (+1 to +6) either on the upper and lower strand stalled the transcription. A nick on either strand located downstream of the abortive initiation region, however, did not stop the elongation, although it appeared to reduce the transcription efficiency to some extent. Furthermore, production of transcripts and efficiency of bypass at a nick highly depended on the location of the nick.