Nucleic Acids Research

Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions (10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures. INTRODUCTION Living organisms have evolved repair mechanisms to eliminate highly lethal double-strand breaks (DSB) from chromosomal DNA (1 for review). Such mechanisms are not only related to homologous recombination pathways (2,3), in which recombinogenic DSB termini interact end-to-strand with duplex DNA (4-7), but also to nonhomologous recombination pathways, in which DSB termini are joined end-to-end (8-11). Nonhomologous DSB repair may be involved in illegitimate recombination pathways, such as chromosomal rearrangements, transposition of transand retroposons or integration of viral DNA into chromosomes (12 and 13 for review). One way to join nonhomologous DSB termini end-to-end would be to shape them to blunt ends (9,14) and ligate them by type I ligase (15). Protruding singlestrands (PSS) created by staggered breakage of duplex DNA could undergo fillin or degradation repair to produce small duplications or deletions at junc©) I R L Press Limited, Oxford, England. 907 Nucleic Acids Research tional flanks (9,16). However, generation of duplications and junctional insertions demonstrate that more complex reaction schemes also exist (14,16). Fortuitious complementarities between short, unrelated DNA-sequences may be called "pseudohomologies" in contrast to "homology", defined as a relation between sequences of common origin. A role of short pseudohomologies in junction formation during nonhomologous joining has been conjectured (reviewed in 17). Direct comparisons between substrate and product DNA sequences unequivocally demonstrates use of pseudohomologies down to singlets as alignment markers in nonhomologous joining reactions (17). Elementary recombination processes in eukaryotes may be studied in vivo by transfer of plasmid or viral DNA into cell systems to induce a strong transient rise of repair and recombination (18-21). Another fruitful approach analyses nonhomologous joining by transfection of linear SV40-DNA with defined ends under conditions selective for joined products (9,11,14,17). To explore nonhomologous joining in vitro we have employed oocytes and eggs of Xenopus laevis, known to be a rich source of ligation and recombination activities. (22-26). We report here that extracts of Xenopus eggs can efficiently join nonmatching DSB termini in various combinations. In some of them the repair system creates ligatable blunt ends by fill-in of PSS ends. However, terminus pairs with PSS of equal polarity are joined by a complex mechanism: noncomplementary PSS ends overlap by a defined number of nucleotides, set by even single pseudohomologous basematches and are maintained during junctional mismatch repair until final ligation. MATERIALS AND METHODS. Extract-preparation from fertilized Xenopus laevis_ggs Artificially fertilized X. laevis eggs prepared according to Newport and Kirshner (27) were extracted within 90 minutes and before cleavage onset. Sorted out, intact, dejellied eggs overlayed with an equal volume of extraction medium (28) were centrifuged in a Beckman SW50.1 Ti-rotor at 30 krpm at 40C for 30 minutes. The interphase (clear plus turbid layer) was removed and thoroughly mixed. Aliquots frozen in liquid N2 and stored at -700C retain activity for up to 6 months. Preparation of substrate DNA Source of substrate DNA was plasmid pSP65 (29) which carries an ampR gene and a polylinker (top panel of Fig.6). In pSP65(Kpn) a KpnI-linker (8-mer, Boehringer, Mannheim) was ligated into the SmaI-site of pSP65 (Fig.6). Plas-