Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

Abstract

DNA damage can significantly modulate expression of the affected genes either by direct structural interference with transcription components or as a collateral outcome of cellular repair attempts. Thus, DNA glycosylases of the base excision repair (BER) pathway have been implicated in negative transcriptional response to several spontaneously generated DNA base modifications, including a common oxidative DNA base modification 8-oxoguanine (8-oxoG). Here, we report that single 8-oxoG situated in the non-transcribed DNA strand of a reporter gene has a pronounced negative effect on transcription, driven by promoters of various strength and with different structural properties, including viral, human, and artificial promoters. We further show that the magnitude of the negative effect on the gene expression correlates with excision of the modified base by OGG1 in all promoter constructs tested. Moreover, by using expression vectors with nuclease resistant backbone modifications, we demonstrate that OGG1 does not catalyse DNA strand cleavage in vivo. Rather, cleavage of the phosphate bond 5′ to 8-oxodG (catalysed by APE1) is essential and universally required for the onset of transcriptional silencing, regardless of the promoter structure. Hence, induction of transcriptional silencing emerges as a ubiquitous mode of biological response to 8-oxoG in DNA.

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

“…For example, when OG is located in recognition elements of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) (1), specificity protein 1 (SP1) (2), or CAMP responsive element binding protein 1 (CREB) (3) transcription factors, protein-binding affinity was significantly reduced. When OG was present in the template strand of proteincoding regions, modest stalling of RNA pol II occurred (4), whereas initiation of DNA repair at OG to yield an abasic site (AP) stopped RNA pol II, leading to truncated transcripts (5). These observations support a hypothesis of OG decreasing gene transcription.…”

8-Oxo-7,8-dihydroguanine, friend and foe: Epigenetic-like regulator versus initiator of mutagenesis

“…1) [11, 33]. Accordingly, we next synthesized catalytically competent, yet stable, AP analogs (tetrahydrofuran, F) in the reporter plasmids and then transfected them into MEF and glioblastoma cells.…”

“…The Hanawalt laboratory found OG in template strands slightly inhibited advancement of RNA pol II and recruits the transcription-coupled repair machinery slowing transcription [10, 47]; additionally, this work concluded that the AP site generated by OGG1 release of OG blocked transcription and initiated transcription-coupled repair. The Khobta laboratory identified that OG is a barrier to transcription when located in either the coding or template strand in a gene coding region [11, 48]. Further, OG is only a transcriptional barrier after conversion to an AP site by OGG1.…”

“…Both TDG binding and removal of 5fC or 5caC and binding and removal of OG by OGG1 yield an AP product (Fig. 1) [11, 57]. The AP product recruits APE1 to continue BER in both cases [57].…”

“…For instance, the presence of OG in template strands can stall the advancement of RNA pol II [10], and initiation of OG repair causes polymerases to stop [11], thus ascribing a role to OG as a transcriptional repressor. However, there are a few notable examples of oxidative stress leading to increased OG formation in the genome in tandem with increased gene expression.…”