NONHOMOLOGOUS RECOMBINATION IN HUMAN-CELLS

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 purif ied human proteins to catalyze nonhomologous end joining in vitro. Ade novirus DNA fragments were shown to be efficiently joined in human cel ls regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nu cleotides 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 abili ty to join test plasmid substrates. Efficient ligation of the linear s ubstrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the jun ction. Substantial purification of the end-joining activity was carrie d out with the human immature T-cell-line HPB-ALL. The protein prepara tion was found to join all types of linear DNA substrates containing h eterologous ends with closely equivalent efficiencies. The in vitro sy stem 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 terme d the NHR ligase. The enriched activity resides in a high-molecular-we ight recombination complex that appears to include and require the hum an homologous pairing protein HPP-1 as well as the NHR ligase. Charact erization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonran domly head-to-head and/or tail-to-tail. The joined ends of the product s 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 majo r NHR pathway for chromosome double-strand break repair.