The ability of Klenow polymerase I, phage T7 polymerase (Sequenase(R))
, human polymerase alpha, and human polymerase beta to synthesize past
(bypass) O-6-methylguanine (O-6-meG) lesions was studied in the prese
nce of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to t
he 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers)
, each containing a single O-6-meG in place of one G in the sequence -
G(1)G(2)CG(3)G(4)T-. Extension products were analyzed by denaturing po
lyacrylamide gel electrophoresis and autoradiography. A fraction of th
e products extended by Klenow fragment terminated either opposite or o
ne base before O-6-meG located at sites 1 and 3. Termination occurred
primarily one base before O-6-meG located at sites 2 and 4. The remain
ing fractions that bypassed the lesions represented full-length produc
t. In control reactions, the O-6-meG-containing templates were anneale
d with complementary 45-mers, repaired with O-6-alkylguanine DNA-alkyl
transferase, annealed with an excess of labeled primer, and extended b
y Klenow fragment. Full-length extension of > 90% was observed with ea
ch template. Primer extension past O-6-meG by DNA polymerase alpha and
Sequenase was partially blocked in a manner which varied with the sit
e of O-6-meG in the template while primer extension by DNA polymerase
beta was completely blocked (< 2% full length extension) with O-6-meG
at sites 1-4, Substitution of MnCl2 for MgCl2 in the reaction mixture
greatly increased the bypass of O-6-meG by Klenow fragment and DNA pol
ymerase alpha but not Sequenase(R) or DNA polymerase beta. The increas
ed ability of Klenow fragment to bypass O-6-meG in the presence of MnC
l2 was found to result from an increased incorporation of G (O-6-meG a
t sites 1 and 2) and A (O-6-meG at sites 1, 2, and 3) opposite the les
ion, The results indicate that O-6-meG can block in vitro polymerizati
on by several DNA polymerases and are consistent with the observed cyt
otoxic effects of methylating agents on mammalian cells.