Replication of O6-Methylguanine-containing DNA by Repair and Replicative DNA Polymerases

Linda J Reha-Krantz,Randy L Nonay,Rufus S Day,Samuel H Wilson

doi:10.1074/jbc.271.33.20088

Linda J Reha-Krantz, Randy L Nonay + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.33.20088

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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.

Highlights

O6-Methylguanine (m6G)1-DNA methyltransferase repairs m6G residues in DNA; 20 –30% of human solid tumor cell lines do not express this repair activity [1]
Previous reports suggested that mammalian pol ␤ could not replicate m6G-containing DNA, but we find that pol ␤ can catalyze trans-lesion replication; the lesion must reside in the optimal context for pol ␤ activity, single- or short nucleotide gapped substrates
Efficient replication of single-nucleotide gaps across from template m6G by partially purified pol ␤ from human glioma cell lines. This result was unexpected from earlier reports of pol ␤ activity on an m6G-containing singlestranded DNA substrate that demonstrated that pol ␤ could not incorporate nucleotides across from m6G [8]

Summary

EXPERIMENTAL PROCEDURES

DNA Polymerases and Accessory Proteins—Recombinant rat pol ␤ was provided by S. The extracts were chromatographed on a Mono Q HR5/5 column (Pharmacia Biotech Inc.) in buffer containing 40 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 10 mM ␤-mercaptoethanol, and a salt gradient from 25 to 500 mM NaCl. Fractions eluting at 240 mM NaCl contained DNA pol ␤ activity as determined by several criteria: high activity on short gapped DNA substrates, but low activity on primed single-stranded DNA templates; and inhibition by dideoxyNTPs, but resistance to aphidicolin. A second set of fractions containing DNA polymerase activity eluted at a higher salt concentration (Ͼ0.4 M NaCl) This polymerase activity was different from pol ␤ activity in the following ways: high activity on long, single-stranded DNA templates compared to short gapped DNA substrates, inhibition by ddNTPs, and sensitivity to aphidicolin. 35 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1.5 mM DTT, 200 ␮g/ml bovine serum albumin, 100 ␮M dNTPs, 6.3% glycerol, 3.75 nM DNA substrate, and 6.25 nM pol ␤, were preincubated for 5 min at 37 °C. The concentration of pol ␤ activity in preparations from human glioma cells was determined by comparing activity with the homogeneously purified rat pol ␤ and the nonmodified gapped DNA substrate

RESULTS

Extension rate dCTP

DNA polymerase

Template G

DISCUSSION