Ak. Basu et al., EFFECT OF SITE-SPECIFICALLY LOCATED MITOMYCIN C-DNA MONOADDUCTS ON INVITRO DNA-SYNTHESIS BY DNA-POLYMERASES, Biochemistry, 32(18), 1993, pp. 4708-4718
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.