Analysis of actively transcribed DNA repair using a transfection-based system

Jean J. Latimer

doi:10.1007/978-1-62703-739-6_37

Analysis of actively transcribed DNA repair using a transfection-based system

Jean J. Latimer

Nova Southeastern University

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

4 Scopus citations

Abstract

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

Original language	English
Title of host publication	Molecular Toxicology Protocols
Editors	Phouthone Keohavong, Kamaleshwar P. Singh, Weimin Gao
Publisher	Humana Press Inc.
Pages	533-550
Number of pages	18
Volume	1105
Edition	3
ISBN (Electronic)	978-1-0716-0223-2
ISBN (Print)	978-1-0716-0222-5, 978-1-0716-0225-6
DOIs	https://doi.org/10.1007/978-1-62703-739-6_37
State	Published - 2014

Publication series

Name	Methods in Molecular Biology
ISSN (Print)	1064-3745

Bibliographical note

Publisher Copyright:
© Springer Science+Business Media New York 2014.

ASJC Scopus Subject Areas

Molecular Biology
Genetics

Keywords

6-4 photoproducts
DNA damage
Global genomic repair (GGR)
Host cell reactivation (HCR)
Luciferase
Nucleotide excision repair (NER)
Thymine dimers
Transcription-coupled repair (TCR)
Transfection
UV irradiation
Cell Line
Humans
Plasmids/genetics
Bacteriophages/genetics
Animals
Ultraviolet Rays
DNA Repair
Luciferases, Firefly/biosynthesis
Genes, Reporter

Disciplines

Molecular Biology
Genetics

Access to Document

10.1007/978-1-62703-739-6_37

Cite this

Analysis of actively transcribed DNA repair using a transfection-based system. / Latimer, Jean J.
Molecular Toxicology Protocols. ed. / Phouthone Keohavong; Kamaleshwar P. Singh; Weimin Gao. Vol. 1105 3. ed. Humana Press Inc., 2014. p. 533-550 (Methods in Molecular Biology).

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

@inbook{992ebb8532be457cb345cae62558f56c,

title = "Analysis of actively transcribed DNA repair using a transfection-based system",

abstract = "Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences. ",

keywords = "6-4 photoproducts, DNA damage, Global genomic repair (GGR), Host cell reactivation (HCR), Luciferase, Nucleotide excision repair (NER), Thymine dimers, Transcription-coupled repair (TCR), Transfection, UV irradiation, Cell Line, Humans, Plasmids/genetics, Bacteriophages/genetics, Animals, Ultraviolet Rays, DNA Repair, Luciferases, Firefly/biosynthesis, Genes, Reporter",

author = "Latimer, \{Jean J.\}",

note = "Publisher Copyright: {\textcopyright} Springer Science+Business Media New York 2014.",

year = "2014",

doi = "10.1007/978-1-62703-739-6\_37",

language = "English",

isbn = "978-1-0716-0222-5",

volume = "1105",

series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

pages = "533--550",

editor = "Phouthone Keohavong and Singh, \{Kamaleshwar P.\} and Weimin Gao",

booktitle = "Molecular Toxicology Protocols",

edition = "3",

}

TY - CHAP

T1 - Analysis of actively transcribed DNA repair using a transfection-based system

AU - Latimer, Jean J.

N1 - Publisher Copyright: © Springer Science+Business Media New York 2014.

PY - 2014

Y1 - 2014

N2 - Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

AB - Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

KW - 6-4 photoproducts

KW - DNA damage

KW - Global genomic repair (GGR)

KW - Host cell reactivation (HCR)

KW - Luciferase

KW - Nucleotide excision repair (NER)

KW - Thymine dimers

KW - Transcription-coupled repair (TCR)

KW - Transfection

KW - UV irradiation

KW - Cell Line

KW - Humans

KW - Plasmids/genetics

KW - Bacteriophages/genetics

KW - Animals

KW - Ultraviolet Rays

KW - DNA Repair

KW - Luciferases, Firefly/biosynthesis

KW - Genes, Reporter

UR - https://www.scopus.com/pages/publications/84908545985

UR - https://www.scopus.com/pages/publications/84908545985#tab=citedBy

U2 - 10.1007/978-1-62703-739-6_37

DO - 10.1007/978-1-62703-739-6_37

M3 - Chapter (peer-reviewed)

C2 - 24623251

AN - SCOPUS:84908545985

SN - 978-1-0716-0222-5

SN - 978-1-0716-0225-6

VL - 1105

T3 - Methods in Molecular Biology

SP - 533

EP - 550

BT - Molecular Toxicology Protocols

A2 - Keohavong, Phouthone

A2 - Singh, Kamaleshwar P.

A2 - Gao, Weimin

PB - Humana Press Inc.

ER -

Analysis of actively transcribed DNA repair using a transfection-based system

Abstract

Publication series

Bibliographical note

ASJC Scopus Subject Areas

Keywords

Disciplines

Access to Document

Other files and links

Fingerprint

Methods for isolating the balbiani body/germplasm from xenopus laevis oocytes

Unscheduled DNA synthesis: The clinical and functional assay for global genomic DNA nucleotide excision repair

Exploring the diagnostic potential of immune biomarker coexpression in Gulf War Illness.

Cite this