O⁶-Methylguanine-induced replication blocks

Abstract

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-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 O6-meG in place of one G in the sequence -G1G2CG3G4T-. 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 O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-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 O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

Replication across O6-Methylguanine by Human DNA Polymerase β in Vitro

“…pol ␤ was previously reported to be unable to bypass m 6 dG (46,47). We have demonstrated that pol ␤ is able to bypass m 6 dG, but at a much reduced efficiency.…”

Chemoprotective gene transfer I: transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro

“…This probably reflects the different mechanisms by which O 6 -chloroethylguanine and with the cytosine of the opposite strand, an event which may be fatal for the cell; 28 the crosslink formation is fairly rapid with a proposed half-life of 6 h. Comparatively less is known about cell killing by O 6 -meG. However, it seems likely that the process of cell killing involves either the mismatch repair system 29 or the induction of chain termination events, 30 both of which are dependent on DNA replication. Therefore, it is a possibility that resynthesis of hATPA before DNA replication may allow sufficient repair of O 6 -meG lesions introduced by temozolomide to prevent cell death.…”

Enhancing hemopoietic drug resistance: A rationale for reconsidering the clinical use of mitozolomide

“…More recently, it has also been shown that O 6 -meG can block DNA polymerase in vitro. 48 If this occurs in vivo, it may also represent a major mode of toxicity for these agents. However, both mechanisms of methylating agent cytotoxicity require DNA replication; thus, lesions may persist in DNA for perhaps up to 36 hours before exerting any biological effect.…”