Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

Abstract

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

“…In this in vitro system, mechanisms of DNA end joining had been previously studied in detail with the help of linear plasmid molecules carrying nonhomologous restriction ends. The features of junction formation were found to be very similar to the ones found in mammalian cells (13)(14)(15)(16). Depending on the terminus configuration to be joined two different joining modes were distinguished in the Xenopus in vitro system: (i) the 'fill-in' mode which preserves PSS sequences in abutting terminus configurations (blunt/PSS; 5'PSS/3'PSS) by fill-in DNA synthesis (21) and (ii) the 'overlap' mode in which antiparallel terminus configurations (5'PSS/5'PSS; 3'PSS/3'PSS) are joined by formation of short mismatched overlap intermediates which are set by single fortuitously matching basepairs and determine the patterns of subsequent repair reactions (22,32).…”

“…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).…”

Illegitimate DNA integration in mammalian cells

“…NHEJ often involves short sequence homology between the joined ends, and additions or deletions of approximately 25 nucleotides or less at the junctions. 34,36,37 Similar processes have been shown to occur during chromosomal double-strand break (DSB) repair, 27,29,38 which again suggests that extrachromosomal and chromosomal repair are mediated by similar cellular machinery.…”

DNA looping by Ku and the DNA-dependent protein kinase

“…This process is capable of preserving the sequences of a variety of end structures (15,17). One model proposed to explain these findings includes an end alignment protein that acts at the break site to allow fill-in synthesis by a polymerase at problematic break sites such as 3Ј overhangs (17).…”

“…Illegitimate recombination joins DNA strands that share little or no sequence homology via an end-to-end joining mechanism (14)(15)(16)(17). This process is capable of preserving the sequences of a variety of end structures (15,17).…”