Abstract
2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (fins= Vmax/Km for insertion) and extension (fext = Vmax/Km for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative fins = 1) ≫ dTMP (2.4 × 10-4) ≈ dAMP (8.1 × 10-5) > dGMP (4.5 × 10-7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative fext = 1) > mFapyG:T (4.6 × 10-3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.
| Original language | English |
|---|---|
| Pages (from-to) | 14589-14597 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 277 |
| Issue number | 17 |
| DOIs | |
| Publication status | Published - Apr 26 2002 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Effects of a guanine-derived formamidopyrimidine lesion on DNA replication. Translesion DNA synthesis, nucleotide insertion, and extension kinetics. / Asagoshi, Kenjiro; Terato, Hiroaki; Ohyama, Yoshihiko; Ide, Hiroshi.
In: Journal of Biological Chemistry, Vol. 277, No. 17, 26.04.2002, p. 14589-14597.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Effects of a guanine-derived formamidopyrimidine lesion on DNA replication. Translesion DNA synthesis, nucleotide insertion, and extension kinetics
AU - Asagoshi, Kenjiro
AU - Terato, Hiroaki
AU - Ohyama, Yoshihiko
AU - Ide, Hiroshi
PY - 2002/4/26
Y1 - 2002/4/26
N2 - 2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (fins= Vmax/Km for insertion) and extension (fext = Vmax/Km for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative fins = 1) ≫ dTMP (2.4 × 10-4) ≈ dAMP (8.1 × 10-5) > dGMP (4.5 × 10-7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative fext = 1) > mFapyG:T (4.6 × 10-3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.
AB - 2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (fins= Vmax/Km for insertion) and extension (fext = Vmax/Km for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative fins = 1) ≫ dTMP (2.4 × 10-4) ≈ dAMP (8.1 × 10-5) > dGMP (4.5 × 10-7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative fext = 1) > mFapyG:T (4.6 × 10-3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.
UR - http://www.scopus.com/inward/record.url?scp=0037177818&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037177818&partnerID=8YFLogxK
U2 - 10.1074/jbc.M200316200
DO - 10.1074/jbc.M200316200
M3 - Article
C2 - 11839760
AN - SCOPUS:0037177818
VL - 277
SP - 14589
EP - 14597
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -