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Nucleic Acids Research, 1994, Vol. 22, No. 9 1643-1650
© 1994

MOLECULAR BIOLOGY

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts

Nicole Beyert+, Susanne Reichenberger, Michael Peters§, Markus Hartung, Bernd Göttlich, Wolfgang Goedecke, Walter Vielmetter and Petra Pfeiffer*

Institut für Genetik der Universität zu Köln Zülpicher Strasse 47, D-50674 Köln, Germany

*To whom correspondence should be addressed

Received January 21, 1994. Revised March 24, 1994. Accepted March 24, 1994.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.

+Present addresses: Institut für Biotechnologie, Forschungszentrum Jülich GmbH, D-52425 Jülich

§Present addresses: Minden Pharma, Karlstrasse 42-44, D-32423 Minden, Germany


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