Replication in vitro and cleavage by restriction endonuclease of 5-formyluracil- and 5-hydroxymethyluracil-containing oligonucleotides.
OBJECTIVE
To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU).


METHODS
The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites. The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined.


RESULTS
The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis. The DNA polymerases incorporated only dAMP opposite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides.


CONCLUSIONS
These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A. It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration.
Zhang
Q M
QM
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Japan.
Sugiyama
H
H
Miyabe
I
I
Matsuda
S
S
Kino
K
K
Saito
I
I
Yonei
S
S
D000818:Animals; D020029:Base Pairing; D045647:DNA Glycosylases; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000737:chemistry; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry