posted on 2020-03-13, 15:24authored bySheng Cao, JohnPatrick Rogers, Jongchan Yeo, Brittany Anderson-Steele, Jonathan Ashby, Sheila S. David
The guanine oxidation
products, 5-guanidinohydantoin (Gh) and spiroiminodihydantoin
(Sp), are mutagenic and toxic base lesions that are removed by Fpg,
Nei, and the Nei-like (NEIL) glycosylases as the first step in base
excision repair (BER). The hydantoins are excellent substrates for
the NEIL glycosylases in a variety of DNA contexts beyond canonical
duplex DNA, implicating the potential impact of repair activity on
a multitude of cellular processes. In order to prepare stable derivatives
as chemical biology tools, oligonucleotides containing fluorine at
the 2′-position of the sugar of 8-oxo-7,8-dihydro-2'-deoxyguanosine2′-F-OG)
were synthesized in ribo and arabino configuration. Selective oxidation
of 2′-F-OG within a DNA oligonucleotide provided the corresponding
2′-F-Gh or 2′-F-Sp containing DNA. The 2′-F-hydantoins
in duplex DNA were found to be highly resistant to the glycosylase
activity of Fpg and NEIL1 compared to the unmodified lesion substrates.
Surprisingly, however, some glycosylase-mediated base removal from
both the 2′-F-ribo- and 2′-F-arabinohydantoin duplex
DNA was observed. Notably, the associated β-lyase strand scission
reaction of the 2′-F-arabinohydantoins was inhibited such that
the glycosylases were “stalled” at the Schiff-base intermediate.
Fpg and NEIL1 showed high affinity for the 2′-F-Gh duplexes
in both ribo and arabino configurations. However, binding affinity
assessed using catalytically inactive variants of Fpg and NEIL1 indicated
higher affinity for the 2′-F-riboGh-containing duplexes. The
distinct features of glycosylase processing of 2′-F-ribohydantoins
and 2′-F-arabinohydantoins illustrate their utility to reveal
structural insight into damage recognition and excision by NEIL and
related glycosylases and provide opportunities for delineating the
impact of lesion formation and repair in cells.