Nonhomologous recombination in human cells.

Abstract

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

“…The majority of the joined products observed by us were consistent with earlier reports (23,43,50). In contrast to some of the earlier reports, we could also see the intramolecular circularization (23,45,51,52). Although, by using T7 exonuclease, we could unambiguously prove formation of circular products (Fig.…”

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).…”

Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

“…In human cells, the filling of PSS ends, as well as the loss of one to several hundred nucleotides, was found in 24 of 25 cases during end joining (6). In fact, Derbyshire et al (6) isolated such an end-joining activity tightly associated with the human homologous-pairing activity and an intrinsic 3Ј-5Ј exonuclease.…”