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"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at
1Departments of Biological Sciences and Chemistry, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, USA.
Abstract:
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.
Insights
Oxidative DNA damage from 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) can cause mutations. Human DNA polymerase beta copying this lesion may also create tandem mutations at adjacent sites.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- 8-Oxo-7,8-dihydro-2 -deoxyguanosine (8-oxo-dG) is a frequent oxidative DNA lesion.
- This lesion promotes G:C to T:A transversion mutations by favoring a syn-conformation and mispairing.
Purpose of the Study:
- To investigate the mutagenic potential of 8-oxo-dG during DNA synthesis by human DNA polymerase beta.
- To determine if DNA polymerase beta introduces errors at template sites adjacent to the 8-oxo-dG lesion.
Main Methods:
- Site-directed mutagenesis to introduce 8-oxo-dG into DNA templates.
- DNA synthesis assays using purified human DNA polymerase beta and alpha.
- Analysis of nucleotide incorporation opposite and adjacent to the 8-oxo-dG lesion.
Main Results:
- DNA polymerase beta showed a slight preference for incorporating dAMP opposite 8-oxo-dG, influenced by sequence context.
- A significant increase in misincorporation (dCMP.A and dGMP.A) was observed at the template site 3 ' upstream of 8-oxo-dG.
- These adjacent errors were sequence-dependent and polymerase-specific, not occurring with DNA polymerase alpha.
- Error rates at adjacent sites were approximately 1% of those opposite 8-oxo-dG, suggesting potential for tandem mutations.
Conclusions:
- Human DNA polymerase beta can introduce tandem mutations during short-gap repair synthesis involving 8-oxo-dG.
- The mutagenic potential of 8-oxo-dG extends beyond the lesion site itself.
- Replacing 8-oxo-dG with 8-bromo-2 -deoxyguanosine reduced misincorporation, highlighting the specific role of oxidative damage.