Opposite stereoselective resistance to digestion by phosphodiesterases I and II of benzo[a]pyrene diol epoxide-modified oligonucleotide adducts.
The deoxyribooligonucleotide 5'-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5'-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3'-OH terminus to the 5'-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5'-->3' direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5'-d(...T-G*...)-3' bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3'-side of the modified G [5'-d(...G*-T...)-3'] is most resistant to digestion by both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mao
B
B
Chemistry Department, New York University, New York 10003.
Li
B
B
Amin
S
S
Cosman
M
M
Geacintov
N E
NE
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005092:Exonucleases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification / Q000378:metabolism*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D012910:Snake Venoms; D013237:Stereoisomerism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012910:Snake Venoms; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D005092:Exonucleases; C022650:spleen exonuclease; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom