Methods - Free-Radical-Induced DNA Damage and Its Repair - page 472

When the Tg lesions is opened by ammonolysis, the resulting product ( α -

R -hydroxy-

exonuclease and the Klenow (exo − ) fragment (Matray et al. 1995; see also Green-
berg and Matray 1997). It is, however, removed by E. coli Fpg and Nth proteins
(Jurado et al. 1998).
A detailed study on the repair of the cA lesion is available (Brooks et al. 2000).
The enzymatic excision of 8-oxo-A by Ogg1 from S. cerevisiae is only effective
when this damage is paired with Cyt (but not Ade, Thy, Gua or Ura; Girard et al.
1998; for a review on the action of this enzyme in excising 8-oxo-G see Nishimura
2002). Substantial neighboring effects are also observed for the excision of other
lesions such as 8-oxo-G or AP sites. Excision of 8-oxo-G by the E. coli Fpg protein
is used as the first step for an improved detection of this lesion (Beckman et al.
2000; ESCODD 2003). There is a large variation in the yields of 8-oxo-G and con-
comitant discussions as to the best method for the detection of this DNA lesion.
Attention has been drawn that incomplete digestion of the damaged DNA by the
enzymatic cocktails typically used may be one of the reasons for such discrepan-
cies, and an improved protocol has been suggested (Huang et al. 2001).
The development in this area of enzymatic action on the various damaged
DNA sites continues to be very active. For this reason, only a very short account
has been given as a kind of f flavor for the reader to see in which direction re-
search in this field seems to expand.

β -ureidoisobutyric acid) inhibits snake venom phosphodiesterase, λ

13. 2 . 3
Detection of DNA Lesions
by Gas Chromatography/Mass Spectrometry

Most of our present knowledge of free-radical-induced DNA lesions is based upon
their identification and often also quantification by GC/MS. In order to convert
the nucleobases and their free-radical-induced products into sufficiently vola-
tile compounds the -NRH and -OH groups have to be trimethylsilylated. Carbo-
hydrate-type products resulting from an alteration of the sugar moiety may be
reduced with NaBH 4 after release or excision from DNA into the corresponding
polyhydric alcohols (Beesk et al. 1979). Reduction with NaBD 4 incorporates a
deuterium atom at the position of a carbonyl function (two deuterium atoms at a
carboxyl group). The mass spectra of the trimethylsilylated polyhydric alcohols
usually allows a firm assignment of their structure when the number of carbon
atoms is known from the GC retention time (Dizdaroglu et al. 1974). A methoxi-
mation of the carbonyl function in combination with a trimethylsilylation of the
hydroxyl groups also converts carbohydrate products into volatile compounds,
and their mass spectra may provide additional information (Dizdaroglu et al.
1977). For the determination of the carbohydrate products, a polyhydric alcohol
that is not formed under the given conditions can be used as internal standard.
For the quantification of the base products, isotopically-labeled reference mate-
rial which also undergoes the various prepurification steps (e.g., by semi-pre-
parative HPLC) may be added (Bianchini et al. 1996; Douki et al. 1996; D'Ham
et al. 1998). The determination of altered bases by GC/MS-SIM (SIM = single-
ion monitoring), after trimethylsilylation, has become the standard method for