Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA.
5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.
Zhang
Q M
QM
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kyoto 606-01, Japan.
Sugiyama
H
H
Miyabe
I
I
Matsuda
S
S
Saito
I
I
Yonei
S
S
D001482:Base Composition; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D009153:Mutagens; D010084:Oxidation-Reduction; D013698:Templates, Genetic; D013941:Thymine / Q000737:chemistry* / Q000378:metabolism; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D009153:Mutagens; D003843:Deoxycytidine Monophosphate; C007777:5-formyluracil; D014498:Uracil; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D013941:Thymine