Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate

Reneau Castore, Cameron Hughes, Austin Debeaux, Jingxin Sun, Cailing Zeng, Shih Ya Wang, Kelly Tatchell, Runhua Shi, Kyung Jong Lee, David J. Chen, Lynn Harrison

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments. The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 2011

Original languageEnglish (US)
Pages (from-to)795-803
Number of pages9
JournalMutagenesis
Volume26
Issue number6
DOIs
StatePublished - Nov 2011

Fingerprint

Bleomycin
Mycobacterium tuberculosis
DNA Damage
Cells
Joining
DNA
Repair
Oncology
Double-Stranded DNA Breaks
Proteins
Fusion reactions
DNA Ligases
Nuclear Localization Signals
Laser damage
Chemotherapy
Radiotherapy
Mutagens
Accessories
Postal Service
Life Cycle Stages

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Castore, R., Hughes, C., Debeaux, A., Sun, J., Zeng, C., Wang, S. Y., ... Harrison, L. (2011). Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate. Mutagenesis, 26(6), 795-803. https://doi.org/10.1093/mutage/ger049

Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate. / Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung Jong; Chen, David J.; Harrison, Lynn.

In: Mutagenesis, Vol. 26, No. 6, 11.2011, p. 795-803.

Research output: Contribution to journalArticle

Castore, R, Hughes, C, Debeaux, A, Sun, J, Zeng, C, Wang, SY, Tatchell, K, Shi, R, Lee, KJ, Chen, DJ & Harrison, L 2011, 'Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate', Mutagenesis, vol. 26, no. 6, pp. 795-803. https://doi.org/10.1093/mutage/ger049
Castore, Reneau ; Hughes, Cameron ; Debeaux, Austin ; Sun, Jingxin ; Zeng, Cailing ; Wang, Shih Ya ; Tatchell, Kelly ; Shi, Runhua ; Lee, Kyung Jong ; Chen, David J. ; Harrison, Lynn. / Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate. In: Mutagenesis. 2011 ; Vol. 26, No. 6. pp. 795-803.
@article{70faf88ba9cf4523a5c03e58d83e393a,
title = "Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate",
abstract = "Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments. The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 2011",
author = "Reneau Castore and Cameron Hughes and Austin Debeaux and Jingxin Sun and Cailing Zeng and Wang, {Shih Ya} and Kelly Tatchell and Runhua Shi and Lee, {Kyung Jong} and Chen, {David J.} and Lynn Harrison",
year = "2011",
month = "11",
doi = "10.1093/mutage/ger049",
language = "English (US)",
volume = "26",
pages = "795--803",
journal = "Mutagenesis",
issn = "0267-8357",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate

AU - Castore, Reneau

AU - Hughes, Cameron

AU - Debeaux, Austin

AU - Sun, Jingxin

AU - Zeng, Cailing

AU - Wang, Shih Ya

AU - Tatchell, Kelly

AU - Shi, Runhua

AU - Lee, Kyung Jong

AU - Chen, David J.

AU - Harrison, Lynn

PY - 2011/11

Y1 - 2011/11

N2 - Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments. The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 2011

AB - Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments. The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 2011

UR - http://www.scopus.com/inward/record.url?scp=80455178850&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80455178850&partnerID=8YFLogxK

U2 - 10.1093/mutage/ger049

DO - 10.1093/mutage/ger049

M3 - Article

VL - 26

SP - 795

EP - 803

JO - Mutagenesis

JF - Mutagenesis

SN - 0267-8357

IS - 6

ER -