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Nucleic Acids Research, 1992, Vol. 20, No. 16 4213-4220
© 1992

MOLECULAR BIOLOGY

Blockage of polymerase-catalyzed DNA chain elongation by chemiscally modified cytosine residues in templates and the release of blockage for readthrough

Tadayoshi Bessho, Noriko Nitta, Kazuo Negishi1 and Hikoya Hayatsu*

Faculty of Pharmaceutical Sciences Tsushima, Okayama 700, Japan 1Gene Research Center, Okayama University Tsushima, Okayama 700, Japan

*To whom correspondence should be addressed

Received May 27, 1992. Revised July 27, 1992. Accepted July 27, 1992.

The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5, 6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisurfite-hydrazlne (and -methoxyamine)- modifled single strand phage-M13mp2 DNA and synthetic oligonucleotldes. Both N4-amino-5,6-dihydrocytoslne- 6-sulfonate and N4-methoxy-5,6-dlhydrocytoslne- 6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites Is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E.coli DNA polymerases in vivo with accompanying errors.


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