O-6-METHYLGUANINE-INDUCED REPLICATION BLOCKS

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 presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 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 polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one 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 remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O-6-meG-containing templates were annealed with complementary 45-mers, repaired with O-6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O-6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site 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 polymerase alpha but not Sequenase(R) or DNA polymerase beta. The increased ability of Klenow fragment to bypass O-6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O-6-meG at sites 1 and 2) and A (O-6-meG at sites 1, 2, and 3) opposite the lesion, The results indicate that O-6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.