Methods - Free-Radical-Induced DNA Damage and Its Repair - page 478

Table 13.1. Enzymes used for the identification of clustered DNA base damage. (Suther-
land et al. 2000)

Enzyme Class recognized Lesion recognized

E. coli Fpg protein (for-
mamidopyrimidine-DNA
glycosylase)

Oxidized purines FAPY-A, FAPY-G, 8-oxo-G, 8-oxo-

A, some abasic sites, to a lesser
extent other modified purines

E. coli Nth protein (endo-
nuclease III)

Oxidized pyrimi-
dines

Ring-saturated or fragmented
Thy residues, e.g. H 2 Thy, Tg, 5-
hydroxy-5-methylhydantoine,
urea, DNA damaged at Gua, some
abasic sites

E. coli Nfo protein (endo-
nuclease IV)

AP sites Several AP sites including oxi-

dized AP sites, urea

13. 2 .14
Detection of SSBs and DSBs with the Help of Supercoiled Plasmids

Plasmids are small pieces of circular supercoiled dsDNA. A SSB causes the plas-
mid to relax into the open circular form, a DSB into the ds linear form. These
there forms can be separated by chromatographic methods (e.g., Bresler et al.
1979). This assay is widely used for studying effects of various agents including
the action of enzymes on damaged DNA.

13. 2 .15
Detection of Clustered Lesions

DNA damage induced by ionizing radiation leads single lesions also to the for-
mation of clustered lesions such as two close-by damaged bases on opposite
strands (Chap. 12). For their detection, DNA is treated with an endonuclease
that induces a SSB at a damaged site. If there are two closely separated lesions
on opposite strands, such treatment induces a DSB which can be detected on a
non-denaturing gel (Sutherland et al. 2000). The enzymes that have been used
and their targets are compiled in Table 13.1.

13. 3
Pulse Radiolysis and Laser Flash Photolysis

The pulse radiolysis technique is close to the better known laser f flash photolysis
(for a monograph see Bensasson et al. 1983). There is one essential difference: in
pulse radiolysis the energy is absorbed by the solvent, e.g., by water in DNA solu-