OGG1-DNA interactions facilitate NF-κB binding to DNA targets

Abstract

DNA repair protein counteracting oxidative promoter lesions may modulate gene expression. Oxidative DNA bases modified by reactive oxygen species (ROS), primarily as 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair (BER) pathway. Because cellular response to oxidative challenge is accompanied by DNA damage repair, we tested whether the repair by OGG1 is compatible with transcription factor binding and gene expression. We performed electrophoretic mobility shift assay (EMSA) using wild-type sequence deriving from Cxcl2 gene promoter and the same sequence bearing a single synthetic 8-oxoG at defined 5′ or 3′ guanine in runs of guanines to mimic oxidative effects. We showed that DNA occupancy of NF-κB present in nuclear extracts from tumour necrosis factor alpha (TNFα) exposed cells is OGG1 and 8-oxoG position dependent, importantly, OGG1 counteracting 8-oxoG outside consensus motif had a profound influence on purified NF-κB binding to DNA. Furthermore, OGG1 is essential for NF-κB dependent gene expression, prior to 8-oxoG excised from DNA. These observations imply that pre-excision step(s) during OGG1 initiated BER evoked by ROS facilitates NF-κB DNA occupancy and gene expression.

Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription

“…Indeed, a rapid up‐regulation of pro‐inflammatory cytokines and chemokines correlated well with an increase in both levels of 8‐oxoG and OGG1 enrichment on the TSS adjacent regulatory sequences, and intriguingly, OGG1 depletion impeded gene expression 25 . In‐depth studies revealed that OGG1's interaction with its DNA substrate facilitated DNA occupancy of sequence‐specific transcription factors on their motifs 33,39,48 . It has been proposed that to prioritize a prompt expression of ROS‐responding genes, OGG1 interacts with DNA without removal of its substrate, which, in turn, facilitates the recruitment of transcription factors and assembly of the transcriptional machinery.…”

“…OGG1 oxidative modification at cysteine residues is a reversible modification that can be regained after redox balance is restored 25,33,36,37 . Inactivation of OGG1 via cysteine oxidation does not interfere with genomic substrate binding, but halts its glycosylase activity 3,23,25,33‐34,39 . The vast numbers of OGG1 K249Q analyses help us to comprehend the mechanism of how oxidatively inactivated OGG1 modulates gene expression.…”

“…Cysteine‐oxidized OGG1 persists its ability to recognize the substrate, bend DNA and extrude the damaged base into the active pocket; however, the conformation of the enzyme/DNA complex does not accommodate for base excision, probably due to the disfavor of the lysine 249 to attack C1′ of the deoxyribose moiety using its ε‐amino group 51 . The biological significance of OGG1 oxidation was proclaimed by studies suggesting that the pre‐excision steps and the associated adjustment of the adjacent DNA by oxidized OGG1 facilitate DNA occupancy of TFD II, Sp1, and NF‐κB, and consequently enhance expression of pro‐inflammatory genes ( TNF , CXCLs , and ILs ) under OS conditions 3,25,33‐34,39,48 . Upon normalization of the cellular redox state, genome fidelity was accomplished via DNA‐BER 33,36,37 .…”

“…Flag‐OGG1 K249Q, Flag‐OGG1 C253A, YFP‐OGG1 K249Q, and YFP‐OGG1 C253A were created using the Fast Mutagenesis System based on Flag‐OGG1 or YFP‐OGG1, respectively. Cxcl2 ‐Luc plasmid including mouse Cxcl2 promoter (−571 to +81) was cloned from the MLE 12 genome and insert into the reporter vector pGL4.2 using restriction enzyme sites Kpn I and Bgl II 25,39 . The plasmid pRL‐SV40 encoding Renilla luciferase driven by an SV40 promoter (Promega) was used as an internal control as described previously 25 .…”

“…However, under the OS condition, OGG1 binding to substrate was not followed by base excision as OGG1 may temporally lose its enzymatic activity due to cysteine oxidation 33,36‐38 . Engagement of transiently inactivated OGG1 with its substrate at the promoter region was associated with recruitment of transcription factors (eg, NF‐κB and Sp1) and RNA polymerase, thereby the transcriptional activation of pro‐inflammatory genes 25,33,39 …”