Nonhomologous DNA end joining in cell‐free systems

Abstract

Double strand DNA breaks are usually caused by ionizing radiation and radiomimetic drugs, but can also occur under normal physiological conditions during double strand break-induced recombination, such as the rearrangement of T-cell receptor and immunoglobulin genes during lymphoid development or the mating type switching in yeast. The main repair mechanism for double strand breaks in higher eukaryotes is nonhomologous DNA end joining (NHEJ), which modifies and ligates the two DNA ends without the help of extensive base± pairing interactions for alignment. Defects in double strand break repair are associated with radiosensitivity, predisposition to cancer and immunodeficiency syndromes, and the analysis of the underlying mutations has lead to the identification of several proteins involved in NHEJ. However, these genetic studies have yielded little information on the mechanism of NHEJ, and while some of the protein factors identified possess the expected enzymatic or DNA-binding activities, the precise role of others remains unclear. Systems for cell-free NHEJ have been available for over 10 years, but the biochemical analysis of NHEJ has lagged behind the genetic analysis, and not a single protein factor required for NHEJ has been identified by biochemical purification and reconstitution of NHEJ activity. Here I review the current status of in vitro systems for NHEJ, summarize the results obtained and information gained, and discuss the outlook for biochemical approaches to study NHEJ.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin

“…We conclude that, in order to cause cell cycle arrest, Vpr requires the presence of helical domains II and III. In most cells, Ku proteins (70 and 80) are located in the nucleus where they play a role in NHEJ, which is involved in DSB repair (Labhart, 1999;de Lange, 2002). Two DSBs on the same DNA molecule lead to the excision of the internal fragment, which can be deleted, inverted or translocated.…”

“…The cell-free NHEJ assay was followed (Labhart, 1999), and nuclear lysates were prepared as previously described (Trojanek et al, 2003). Briefly, NHEJ reactions were performed in the following conditions: 50 mg of nuclear lysate; 1 mM ATP; 0.25 mM dNTPs; 25 mM Tris-acetate (pH 7.5); 100 mM potassium acetate; 10 mM magnesium acetate; and 1 mM dithiothreitol.…”

BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species–dependent DNA double-strand breaks

“…pBluescript plasmid linearized by XhoI ϩ XbaI digestion creating noncomplementary 5' overhangs was used as the substrate to assess the activity of NHEJ, which generates the products containing multimers of plasmid. 30,45 The substrate was added to cell lysates from 32Dcl3 parental, BCR/ABL wild-type, and BCR/ABL[K1172R] kinase-inactive (BCR/ABL [kinϪ] ) cells, and NHEJ products were analyzed by agarose gel electrophoresis. BCR/ABL kinase activity was responsible for more than a 5-fold increase of NHEJ activity; inactivation of the kinase by point mutation (K1172R) reduced NHEJ by 3-fold ( Figure 3B).…”

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

“…The cell-free NHEJ assay was followed, 35 and nuclear lysates were prepared according to the protocol previously described. 32 Briefly, NHEJ reactions were performed in the following conditions: 50 lg of nuclear lysate; 1 mM ATP; 0.25 mM dNTPs; 25 mM Tris-Acetate (pH 7.5); 100 mM potassium acetate; 10 mM magnesium acetate and 1 mM DTT.…”