The base-pairing properties of O⁶-methylguanine during DNA replication in vitro

Author

Elizabeth T. Snow

Date of Award

12-1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Sankar Mitra

Committee Members

Julian Preston, Peter Lalley, Bruce Jacobson, Warren Masker

Abstract

O⁶-methylguanine (m⁶G), because of its ability to "mispair" with thymine during replication, is an important premutagenic lesion produced in DNA by simple alkylating carcinogens. The kinetics of incorporation of nucleotide precursors opposite m⁶G were analyzed during replication of synthetic deoxynucleotide polymers containing m⁶dG as the only modified base by T5 phage-induced DNA polymerase. Incorporation of dTMP opposite m⁶dG. dTTP and dCTP are linear competitive inhibitors for incorporation opposite m⁶dG. dAMP is also incorporated opposite m⁶dG but does not compete with dTMP incorporation. It is proposed that dA base-pairs with m⁶dG in the dA_anti:m⁶dG_syn configuration. Different DNA polymerases vary in their ability to discriminate between dT:m⁶dG and dC:m⁶dG base-pairs. The relative number of mutations produced during replication of DNA containing m⁶dG will depend on the DNA polymerase, DNA precursor pool concentrations, DNA sequence, and possibly other factors.

Alkylated nucleotides in the DNA precursor pool may also contribute to mutagenesis induced by simple alkylating agents. M⁶dGMP can be incorporated into DNA by several different DNA polymerases using both natural and synthetic template-primers. The rate of incorporation of m⁶dGMP is significantly less than the rate of incorporation of normal dNTP substrates. Incorporation of m⁶dGMP during replication of poly(dA-dT) is inhibited by dATP but not dTTP. Different polymerases differ in their ability to utilize m⁶dGTP as a substrate during DNA replication. Incorporation of m⁶dGMP from the nucleotide pool will depend on the DNA polymerase, deoxynucleotide pool concentrations, and other possible factors. Mutagenesis due to m⁶dGMP incorporation during DNA replication in vivo will be insignificant compared to mutagenesis due to the m⁶dG produced in DNA directly by simple alkylating carcinogens.

Recommended Citation

Snow, Elizabeth T., "The base-pairing properties of O⁶-methylguanine during DNA replication in vitro. " PhD diss., University of Tennessee, 1982.
https://trace.tennessee.edu/utk_graddiss/13325