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Methods Enzymol. 2017;591:287-306. doi: 10.1016/bs.mie.2017.03.014. Epub 2017 May 3.

Strategies and Methods of Transcription-Coupled Repair Studies In Vitro and In Vivo.

Author information

1
New York University School of Medicine, New York, NY, United States.
2
New York University School of Medicine, New York, NY, United States; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, United States.
3
New York University School of Medicine, New York, NY, United States; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, United States. Electronic address: evgeny.nudler@nyumc.org.

Abstract

Transcription-coupled repair (TCR) serves an important role in preserving genome integrity and maintaining fidelity of replication. Coupling transcription to DNA repair requires a coordinated action of several factors, including transcribing RNA polymerase and various transcription modulators and repair proteins. To study TCR in molecular detail, it is important to employ defined protein complexes in vitro and defined genetic backgrounds in vivo. In this chapter, we present methods to interrogate various aspects of TCR at different stages of repair. We describe promoter-initiated and nucleic acid scaffold-initiated transcription as valid approaches to recapitulate various stages of TCR, and discuss their strengths and weaknesses. We also outline an approach to study TCR in its cellular context using Escherichia coli as a model system.

KEYWORDS:

Nucleotide excision repair; NusA; RNA polymerase; Transcription-coupled DNA repair; UvrD; ppGpp

PMID:
28645373
DOI:
10.1016/bs.mie.2017.03.014
[Indexed for MEDLINE]

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