The influence of the exocyclic pyrimidine 5-methyl group on DNAse I cleavage and sequence recognition by drugs.
Incorporation of modified nucleotides into DNA, using the PCR, has allowed us to probe the influence that the exocyclic 5-methyl group of pyrimidines has on DNAse I cleavage and sequence recognition by drugs. The results show that removal of the methyl group from the major groove, made possible by substituting uridine for thymidine, allows DNAse I to cleave more readily at AT-rich regions compared to normal DNA. By contrast, addition of an extra methyl group, contrived by substituting 5-methylcytidine for normal cytidine, allows DNAse I to cleave more readily at GC-rich regions compared to normal DNA. In the cutting pattern of DNA containing both uridine and 5-methyl cytosine, we find the cleavage characteristics of both the single-substituted DNA species combined. Thus, the presence or absence of the exocyclic 5-methyl group in the major groove has a strong influence on the relative intensity of cleavage of phosphodiester bonds by DNAse I. These nucleotide substitutions can also influence the sequence-selective binding of drugs to DNA. Whereas removal of the methyl group (replacement of T with U) generally has little effect on sequence recognition by a variety of drugs, addition of a methyl group (replacement of C with M) generates new binding sites for some intercalators, namely daunomycin, DACA and SN16713.
Minnock
A
A
Department of Pharmacology, University of Cambridge, UK.
Crow
S
S
Bailly
C
C
Waring
M J
MJ
D001345:Autoradiography; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D017931:DNA Primers; D003630:Daunorubicin / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011743:Pyrimidines / Q000378:metabolism*
D017931:DNA Primers; D011743:Pyrimidines; D004247:DNA; D003850:Deoxyribonuclease I; C030986:pyrimidine; D003630:Daunorubicin