The identification and characterization of mammalian proteins involved in the rejoining of DNA double-strand breaks in vitro

Using a combination of specific assays and biochemical fractionation of mammalian extracts, we have identified multiple activities involved in the rejoining of DNA double-strand breaks. Fractionation of whole cell extracts from calf thymus has identified four biochemically distinct fractions capable of joining double-strand breaks, and an activity Rejoin Enhancement Protein (REP-1), that stimulates this process. We also show that REP-1 directly stimulates a DNA ligase and that this stimulation is associated with the increased turnover of the adenylated intermediate formed by all ATP-dependent DNA ligases. Activity relationships between the rejoining fractions and REP-I indicates that the joining of double-strand breaks is carried out by protein complexes of which REP-1 is a component. In support of this, the cellular activities identified here that can efficiently rejoin double-strand breaks, do not show detectable adenylation products. Western analysis also shows that several proteins that have been suggested to be involved in the joining of double-strand breaks, such as the Ku heterodimer, are not present in all fractions that contain rejoining activity. These data strongly suggests that many different activities exist that can rejoin double-strand breaks and that this process is not dependent on the presence of proteins such as the end-binding protein Ku.