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Nucleic Acids Research, 1981, Vol. 9, No. 18 4755-4766
© 1981

MOLECULAR BIOLOGY

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA

Yasunori Machida1,*, Tuneko Okazaki1,**, Tetsuo Miyake2, Eiko Ohtsuka2 and Morio Ikehara2

1Institute of Molecular Biology, Faculty of Science, Nagoya University Nagoya, Japan 464 2Faculty of Pharmaceutical Sciences, Osaka University Osaka, Japan 565

**To whom correspondence should be addressed.

Received May 11, 1981.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d{pRib[32P]poly(T)} and d{Rib[32P]poly(T)}. Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escheriohia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.

*Present address: Department of Microbiology, School of Medicine, State University of New york at Stony Brook, New York 11794


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