Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA.
We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group). It was observed that the enzyme acted only on the latter isomer. Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase [Weinfeld, M., Liuzzi, M., & Paterson, M. C. (1989) Nucleic Acids Res. 17, 3735-3745], we were able to devise a postlabeling assay for these biologically important DNA lesions. The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites. Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour. This value compares favorably with previously published data of others, acquired by alternative approaches. The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour.
Weinfeld
M
M
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Liuzzi
M
M
Paterson
M C
MC
D001080:Apurinic Acid; D001483:Base Sequence; D004247:DNA; D003850:Deoxyribonuclease I; D015226:Dinucleoside Phosphates; D004926:Escherichia coli / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*
D015226:Dinucleoside Phosphates; D009838:Oligodeoxyribonucleotides; C027903:oligo (dT); D001080:Apurinic Acid; D004247:DNA; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D003850:Deoxyribonuclease I