Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3',5')-deoxyadenosine photoproduct.
Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhao
X
X
Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Kao
J L
JL
Taylor
J S
JS
D001483:Base Sequence; D004247:DNA / Q000528:radiation effects*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010777:Photochemistry; D013698:Templates, Genetic; D013936:Thymidine / Q000737:chemistry*; D014466:Ultraviolet Rays
D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D013936:Thymidine