EFFECT OF SITE-SPECIFICALLY LOCATED MITOMYCIN C-DNA MONOADDUCTS ON INVITRO DNA-SYNTHESIS BY DNA-POLYMERASES

A series of site-specifically modified oligodeoxynucleotides were synt hesized that contained either of the two known mitomycin C-DNA monoadd ucts. In vitro DNA synthesis was carried out on some of these template s using a modified bacteriophage T7 DNA polymerase (Sequenase), AMV re verse transcriptase, and two different varieties of Escherichia coli D NA polymerase I (Klenow fragment)-one that carries the normal 3' --> 5 ' exonuclease activity and a mutant protein that lacks this enzymatic function. Regardless of the type of DNA polymerase being used, DNA syn thesis was terminated nearly quantitatively at the nucleotide 3' to ea ch of these two monoadduct sites, although primer extension to full le ngth of the template was noted with the unmodified control template. S ubstitution of Mn2+ for Mg2+ at a high concentration of the deoxynucle otide triphosphates resulted in incorporation of nucleotides opposite the adduct in the incubations with Sequenase or the 3' --> 5' exonucle ase-free Klenow fragment; however, primer extension beyond the adduct site did not take place. These studies demonstrated that the mitomycin monoadducts are strong blocks of replication and are likely to be tox ic lesions in vivo. Since previous molecular modeling studies and mole cular mechanical calculations indicated that the mitomycin adduction d oes not induce severe distortions at the site of adduction, a lack of base-pairing ability of the modified base in the extended product is u nlikely to be the reason for the inhibitory effect. Instead, energy-mi nimized structural models indicated that additional hydrogen-bonding i nteractions have been introduced by the mitomycin moiety, and perhaps this increased thermodynamic stabilization of a distorted structure of the replication fork, in turn, may block the replication bypass. Expe rimental evidence of increased thermodynamic stability was provided by thermal melting of a template/primer complex that presumably a polyme rase encounters in a typical replication fork. Consistently higher T(m ) of the adducted ''replication fork'' was noted when compared to its unmodified counterpart.