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Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases
L J Reha-Krantz1, R L Nonay, R S Day
1Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Abstract:
The biological consequences of O6-methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.
Insights
O6-methylguanine (m6G) in DNA is mutagenic, but some DNA polymerases can replicate past it. This study shows mammalian pol beta and T4 DNA polymerase can perform trans-lesion replication of m6G, with dTMP incorporation being rate-limiting.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- O6-methylguanine (m6G) is a DNA lesion that causes cytotoxicity and mutations during replication.
- Cellular tolerance to m6G suggests some DNA polymerases can bypass this lesion.
- Previous studies indicated mammalian pol beta cannot replicate m6G-containing DNA.
Purpose of the Study:
- To investigate the ability of mammalian pol beta and bacteriophage T4 DNA polymerase to replicate DNA containing O6-methylguanine.
- To determine the substrate requirements and limitations for trans-lesion replication of m6G by these polymerases.
Main Methods:
- In vitro replication assays using DNA templates containing O6-methylguanine.
- Comparison of replication efficiency and fidelity across different DNA polymerase and substrate contexts.
- Investigation of the role of accessory proteins, such as T4 gp45, in polymerase activity.
Main Results:
- Mammalian pol beta can perform trans-lesion replication of m6G, particularly in single- or short nucleotide gapped substrates.
- Primed single-stranded DNA templates were poor substrates for pol beta, consistent with prior reports.
- Bacteriophage T4 DNA polymerase, with the gp45 accessory protein, efficiently replicated m6G in primed single-stranded DNA templates.
- Incorporation of deoxythymidine monophosphate (dTMP) opposite the m6G lesion was the rate-limiting step for both polymerases.
Conclusions:
- Mammalian pol beta possesses trans-lesion replication capabilities for O6-methylguanine under specific substrate conditions.
- T4 DNA polymerase demonstrates robust replication of m6G-containing DNA, highlighting the importance of accessory proteins.
- The preferential incorporation of dTMP opposite m6G is a conserved feature and a bottleneck in the replication of this lesion by different DNA polymerases.