Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Abstract

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

“…Extensive analysis of end-joining reactions of plasmid DNA have demonstrated that short sequence homologies at the termini play a role in the repair of extrachromosomal DSBs in vivo (44) and in DNA end joining as tested in cell extracts (35,39). Our data support the relevance of these studies to the repair of chromosomal DSBs.…”

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.…”

Nonhomologous DNA end joining of synthetic hairpin substrates inXenopus laevisegg extracts

“…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).…”

“…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).…”