Article
Construction and purification of site-specifically modified DNA templates for transcription assays

Authors: Perlow R.A.; Schinecker T.M.; Kim S.J.; Geacintov N.E.; Scicchitano D.A.

Source: Nucleic Acids Research, Volume 31, Number 7, 1 April 2003 , pp. e40-e40(1)

Publisher: Oxford University Press

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Abstract:

Chemical and physical agents can alter the structure of DNA by modifying the bases and the phosphate–sugar backbone, consequently compromising both replication and transcription. During transcription elongation, RNA polymerase complexes can stall at a damaged site in DNA and mark the lesion for repair by a subset of proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Although our understanding of DNA lesion effects on transcription elongation and the associated effects of stalled transcription complexes on DNA repair is broadening, the attainment of critical data is somewhat impeded by labor-intensive, time- consuming processes that are required to prepare damaged DNA templates. Here, we describe an approach for building linear DNA templates that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II. The method is rapid, making use of biotin–avidin interactions and paramagnetic particles to purify the final product. Data are supplied demonstrating that these templates support transcription, and we emphasize the potential versatility of the protocol and compare it with other published methods.

Document Type: Original article

Affiliations: Departments of Biology and Chemistry, New York University, 100 Washington Square East, New York, NY 10003, USA

Publication date: 2003-04-01

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Nucleic Acids Research (NAR) is a fully Open Access journal, providing rapid publication of leading edge research into the nucleic acids under the following categories: chemistry, computational biology, genomics, molecular biology, nucleic acid enzymes, RNA and structural biology. There is a Survey and Summary section, and methods papers are published
in NAR Methods Online. Each year the first issue is devoted to biological databases, and a later issue to relevant web-based software resources.