Hypoxanthine\xanthine oxidase\Fe3+\ethylenediaminetetraacetate (EDTA) was u
sed to modify ss M13 mp18 phage DNA. The dominant base modifications found
by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol.
Analysis of in vitro DNA synthesis on oxidatively modified template by thr
ee DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of p
olymerase I from Escherichia coli were blocked mainly by oxidized pyrimidin
es in the template whereas some purines that were easily bypassed by the pr
okaryotic polymerases constituted a block for DNA polymerase beta from calf
thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde
content increased 16-fold on oxidation, yielded a final product with a dis
crete ladder of premature termination bands. When DNA synthesis was perform
ed on template from which FapyAde, FapyGua, and 8OHGua were excised by the
Fpg protein new chain terminations at adenine and guanine sites appeared or
existing ones were enhanced. This suggests that FapyAde, when present in D
NA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depen
ds on the sequence context and DNA polymerase. FapyGua might possess simila
r properties. (C) 2000 Elsevier Science Inc.