J. Biol. Chem., Vol. 259, Issue 24, 15069-15077, 12, 1984
Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines
MP Merville, J Piette, M Lopez, J Decuyper and A van de Vorst
Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were
primed either with restriction fragments or synthetic primers and
irradiated with near UV light in the presence of promazine derivatives.
These DNAs were used as template for in vitro complementary chain synthesis
by Escherichia coli DNA polymerase I large fragment. Chain terminations
were observed by denaturing polyacrylamide gel electrophoresis of the
synthesis products and localized by comparison with a standard dideoxy
sequencing pattern. More than 90% of the chain terminations were mapped
exactly one nucleotide before a guanine residue. In addition, photoreaction
was shown to occur more predominantly with guanine residues localized in
single-stranded parts of the genome. The same guanine residues could also
be damaged when the reaction was performed, in the dark, in the presence of
the artificially generated promazine cation radicals. Using the BamHI-SmaI
adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a
covalent addition of the promazine, or of a cation radical photodegradation
product, on the guanine moiety. Kinetics of chlorpromazine photoaddition on
single-stranded and double-stranded DNAs were determined.
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