Methods - Free-Radical-Induced DNA Damage and Its Repair - page 471

Although 35% HF in pyridine is considered to be milder than the formic acid
treatment, HMUra is degraded under such conditions (Douki et al. 1996). More-
over, 60% formic acid is believed to be a stronger hydrolyzing agent as compared
to 88% formic acid due to the higher degree of dissociation. Yet, the final yield
of HMUra (in the plateau region after 2 h) is significantly lower in the case of the
lower formic acid concentration (Douki et al. 1996). Thus, there is no ideal con-
dition, and for each DNA lesion special conditions may have to be worked out to
avoid major artifacts (Cadet et al. 1997a). Apparently, the discussion on potential
artifacts continues (Dizdaroglu 1998) and experimental expertise acquired over
the years in one laboratory is difficult to transfer to another one.
13.2.2
Excision by Enzymatic Hydrolysis
Glycosylases excise damaged nucleobases (for a review see Krokan and Standal
1997). They are grouped into 'simple glycosylases' that do not form DNA strand
breaks and 'glycosylases/abasic site lyases' that concomitantly induce a stand
break (Dodson and Lloyd 2002). This may have to be taken into account when
using these enzymes for the detection of damaged sites (for the use of endonucle-
ases for fingerprinting of DNA damage, see Epe and Hegler 1994).
As mentioned brief ly above, the enzymatic excision of damaged nucleobases
may cause some problems. A case in point is the action of nuclease P1. While a
single 8-oxo-G lesion is excised as the damaged nucleoside, the clustered 8-oxo-
G/Fo lesion is only obtained as modified dinucleotide (Maccubbin et al. 1992).
Another example is the hydrolysis of dG*pC which severely inhibits the action of
bovine spleen phosphodiesterase, while HMUrapA shows only very little inhibi-
tion (Maccubbin et al. 1991). Enzymatic hydrolysis of DNA is, in fact, the recom-
mended method for the determination of HMUra (Teebor et al. 1984; Frenkel et
al. 1985). It is recalled that mammalian cells cope with this DNA lesion with the
help of a hydroxymethyluracil glycosylase (Hollstein et al. 1984).