Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction.
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 (K(d) = ∼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.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (UTMB), Galveston, Texas 77555-1079, USA.
Muralidhar L
ML
Hegde
Srijita
S
Banerjee
Pavana M
PM
Hegde
Larry J
LJ
Bellot
Tapas K
TK
Hazra
Istvan
I
Boldogh
Sankar
S
Mitra
D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D057809:HEK293 Cells; D034722:Heterogeneous-Nuclear Ribonucleoprotein U / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D011485:Protein Binding
D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D045647:DNA Glycosylases; C473634:NEIL1 protein, human