The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro

Abstract

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impede transcription, which could make them viable substrates for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting nominal lesion bypass of 1.6 ± 0.5%. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR since each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair–π and CH–π interactions

“…S1). The strong Gh-induced stalling is also consistent with a previous report using HeLa nuclear extract (23). For both R-Sp and S-Sp lesions, ATP and GTP incorporation are preferred and CTP incorporation is greatly inhibited in the first step.…”

“…In addition, Sp and Gh lesions cause replication blocks by phage DNA polymerase and lead to slow misincorporation of A or G by reverse transcriptases (18)(19)(20). As for transcription, in sharp contrast to effective transcriptional bypass of 8OG (1,21,22), an early study using HeLa nuclear extract reported that the presence of Gh or Sp in templatestrand DNA (tsDNA) causes severe transcriptional stalling (23). However, it is not clear how these nonbulky lesions (much smaller size in comparison with guanine and other bulky lesions) lead to strong stalling.…”

Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis

“…Other indirect roles for unrepaired 8-oxoGs in provoking telomere defects are possible, but our data suggest that they likely play a minor role, if any. First, although 8-oxoGs can be converted to replication-blocking hydantoin lesions (Henderson et al, 2003;Kolbanovskiy et al, 2017), we did not detect recruitment of the hydantoin repair enzyme NEIL1 glycosylase (Wallace, 2013). Second, 8-oxoG could interfere with TRF1 or TRF2 binding directly (Opresko et al, 2005) or by causing G to T mutations because 8-oxoG miscodes for A (Hsu et al, 2004;Markkanen, 2017).…”

The impact of oxidative DNA damage and stress on telomere homeostasis

“…Since 8-oxoG has a lower redox potential than the normal unmodified bases, it can be further oxidized to other lesions including spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) (Luo et al, 2001). These distorting hydantoin lesions impede DNA replication and transcription (Henderson et al, 2003; Kolbanovskiy et al, 2017), but can be removed by any of the three NEIL glycosylases (reviewed in (Wallace, 2013)). Interestingly, NEIL1 and mNeil3 remove Sp and Gh from telomeric G-quadruplex structures and ssDNA, whereas 8-oxoG cannot be removed (Zhou et al, 2013).…”