Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction

Muralidhar L. Hegde, Srijita Banerjee, Pavana M. Hegde, Larry J. Bellot, Tapas Hazra, Istvan Boldogh, Sankar Mitra

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (Kd = ∼54 nM). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

Original languageEnglish (US)
Pages (from-to)34202-34211
Number of pages10
JournalJournal of Biological Chemistry
Volume287
Issue number41
DOIs
StatePublished - Oct 5 2012

Fingerprint

Heterogeneous-Nuclear Ribonucleoprotein U
DNA Repair
Repair
DNA
Oxidative stress
Proteins
DNA Glycosylases
Oxidative Stress
Genes
Cells
Cytoprotection
Single-Stranded DNA
Human Genome
Circular Dichroism
Oxidants
Byproducts

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction. / Hegde, Muralidhar L.; Banerjee, Srijita; Hegde, Pavana M.; Bellot, Larry J.; Hazra, Tapas; Boldogh, Istvan; Mitra, Sankar.

In: Journal of Biological Chemistry, Vol. 287, No. 41, 05.10.2012, p. 34202-34211.

Research output: Contribution to journalArticle

Hegde, Muralidhar L. ; Banerjee, Srijita ; Hegde, Pavana M. ; Bellot, Larry J. ; Hazra, Tapas ; Boldogh, Istvan ; Mitra, Sankar. / Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 41. pp. 34202-34211.
@article{42cff8e7df234fefa13b61785fb646d0,
title = "Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction",
abstract = "Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (Kd = ∼54 nM). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.",
author = "Hegde, {Muralidhar L.} and Srijita Banerjee and Hegde, {Pavana M.} and Bellot, {Larry J.} and Tapas Hazra and Istvan Boldogh and Sankar Mitra",
year = "2012",
month = "10",
day = "5",
doi = "10.1074/jbc.M112.384032",
language = "English (US)",
volume = "287",
pages = "34202--34211",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "41",

}

TY - JOUR

T1 - Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction

AU - Hegde, Muralidhar L.

AU - Banerjee, Srijita

AU - Hegde, Pavana M.

AU - Bellot, Larry J.

AU - Hazra, Tapas

AU - Boldogh, Istvan

AU - Mitra, Sankar

PY - 2012/10/5

Y1 - 2012/10/5

N2 - Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (Kd = ∼54 nM). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

AB - Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (Kd = ∼54 nM). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

UR - http://www.scopus.com/inward/record.url?scp=84867261398&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84867261398&partnerID=8YFLogxK

U2 - 10.1074/jbc.M112.384032

DO - 10.1074/jbc.M112.384032

M3 - Article

VL - 287

SP - 34202

EP - 34211

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 41

ER -