Abstract
5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.
Highlights
5-Formyluracil, a major methyl oxidation product of thymine, forms correct and incorrect base pairs during DNA replication
Chem. 274, 25136 –25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II)
Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS
Summary
Chemicals—Ultra-pure dNTPs were purchased from Amersham Pharmacia Biotech. 5-Formyl-2Ј-deoxyuridine (fdU) and 5-formyl-2Јdeoxyuridine 5Ј-triphosphate (fdUTP) were synthesized and purified as described [4]. 25T and 25FU containing thymine and fU at the same position, respectively, were prepared by DNA polymerase reactions using template/primer 30A/P1 as described in the accompanying paper [5] and separated from the complementary strand 30A by preparative 16% polyacrylamide gel electrophoresis under denaturing conditions. The substrates (2 nM) were preincubated with MutS protein (0.049 – 0.39 g, approximately 0.5– 4 pmol) in Buffer B (9 l) at 0 °C for 10 min, and AlkA (10 ng) diluted in Buffer B (1 l) was added to the reaction mixture. Binding reactions were performed by mixing 3 l of 5Ј-labeled oligonucleotide duplexes (25FU/30A, 25FU/30G, 25T/30A, and 25T/30G, 0.1 pmol, Table I) in Buffer D with 2 l of MutS protein (0.2– 0.82 g, approximately 2.1– 8.4 pmol) appropriately diluted in Buffer E. Free and bound DNA species were quantitated by Fuji BAS 2000
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