Mechanisms of overlap formation in nonhomologous DNA end joining.

Abstract

Rejoining of nonhomologous DNA termini plays a central role in processes of illegitimate recombination. In Xenopus egg extracts, DNA ends with noncomplementary 4-nucleotide antiparallel single-strand protrusions are assumed to bejoined by formation of short mismatched overlap intermediates. The extents of these overlaps may be set by single fortuitously matching base pairs and determine the patterns of subsequent gap filling and nick ligation. Under conditions of alternative overlap settings, rules for the most probable joining pathway and the effects of mismatches on junction formation were analyzed. We show that in certain cases, fill-in and ligation converting overlap intermediates into covalently closed junctions may proceed in the presence of unrepaired mismatches, whereas in other cases, completion of junction formation is preceded by removal of mismatches. Results are discussed in relation with "alignment" proteins postulated to structurally support overlap heteroduplexes during junction formation.

Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

“…The junction resulting from joining of the 5'-hairpin combination B-72/S-82 was sujete %t diex-sqec ngAn verirfied to cornt-ain the sequence (5'-GATCGA) which is expected to arise by overlap formation (compare Fig.2A). From these data we conclude that the joining of hairpin substrates proceeds by the same mechanisms as previously described for the joining of plasmid molecules (22). Joining of hairpins with blunt ends Hairpins with blunt ends were prepared from unlabelled 5'-hairpins by fill-in of the 5'-Bam or Sal-PSS with Klenow polymerase.…”

“…1). The nonhomologous Bam/Sal terminus configuration was chosen because it had been previously shown to be efficiently joined in the Xenopus in vitro system (20,22).…”

“…Mechanisms of DNA end joining have been investigated in detail in a variety of eucarytic in vivo (cultured mammalian cells, [13][14][15][16]; Xenopus laevis eggs, 17; yeast, 9,18,19) and in vitro systems (extracts from Xenopus laevis eggs [20][21][22]; nuclear extracts from human cells, [23][24][25][26][27] all of which were shown to be able to join DNA termini containing either blunt ends or short protruding single strands (PSS). Junctional sequence analysis revealed the existence of various different junction types with distinct features which led to the postulation of different pathways of DNA end joining (21,22,25,26,27).…”

“…Junctional sequence analysis revealed the existence of various different junction types with distinct features which led to the postulation of different pathways of DNA end joining (21,22,25,26,27).…”

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

“…Obviously, free DMA ends are not just repaired by DMA polymerase and then ligated. It was concluded that the DNA ends are juxtaposed by some cellular factor (Thode et al, 1990;Pfeiffer et al, 1994a;Pfeiffer et al, 1994b;Reichenberger et al, 1996) because the repair polymerase can also copy across two unligatable single-strands. It remains to be seen whether this so-called alignment factor(s) is related to the apparatus that performs site-directed recombination of immunoglobulin and T-cell receptor genes (reviewed in Jackson and Jeggo, 1995;Weaver, 1995).…”