Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

Abstract

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G 1 /early S phase and again at the G 2 phase in wild-type cells. Interestingly, only the deficit of the G 1 /early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G 2 phase may be required for exit from a DNA damage-induced G 2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G 2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.Normal lymphoid development is marked by the somatic rearrangement of separate genetic elements to form functional immunoglobulin and T-cell receptor genes. This process, known as V(D)J recombination, is mediated by a site-specific DNA rearrangement mechanism that targets conserved signal sequences flanking the genetic elements (reviewed in reference 46). The initial steps of V(D)J recombination involve the recognition of the signal sequences and the introduction of DNA double-strand breaks (DSBs) adjacent to them. These steps are carried out by the products of two lymphoid-specific genes, RAG-1 and RAG-2 (20, 51).Several studies have suggested that V(D)J recombination events may be restricted during the cell cycle. Analysis of RAG-2 protein demonstrated that it accumulated in cells preferentially in the G 0 /G 1 phase of the cell cycle, declined more than 20-fold before entering the S phase, and remained low throughout the S, G 2 , and M phases (47). Interestingly, the levels of RAG-1 protein, which fluctuated to a much lesser extent, still declined fivefold in the S phase (48). Lastly, the double-strand signal sequence breaks associated with V(D)J recombination can be detected only in the G 0 /G 1 phase of the cell cycle (65). These data strongly imply that the initiation of V(D)J recombination is mostly, and perhaps entirely, restricted to the G 1 phase of cycling cells.While RAG-1 and RAG-2 control the initial steps of V(D)J recombination, the later steps appear to be controlled by genes involved in generalized DNA DSB repair. In particular, a complex, DNA-dependent protein kinase (DNA-PK), which has DNA-dependent serine-threonine protein kinase activity and which consists of at least three components, the 465-kDa catalytic subunit (DNA-PK cs ) and the heterodimeric Ku protein, has been shown to be intimately involved in generalized DNA D...

DNA Protein Kinase–Dependent G2 Checkpoint Revealed following Knockdown of Ataxia-Telangiectasia Mutated in Human Mammary Epithelial Cells

“…In line with these results, inhibition of DNA-PK by DNA-PK-specific inhibitors also caused G 2 -M accumulation in response to irradiation and topoisomerase II poisons (46,47) and DNA-PKcs-deficient cells were found to accumulate in G 2 -M after IR (48). It is interesting that these studies showed that loss of DNA-PK led to G 2 -M accumulation in response to DNA damage (45)(46)(47). It was concluded that DNA-PKcs-deficient cells had an intact IR-induced G 2 checkpoint (48).…”

“…Gladdy et al (44) further showed that, in fact, scid/scid, ATM À/À embryos failed to undergo normal organogenesis and this was due to increased p53-independent apoptosis. In addition, Lee et al (45) reported that DNA-PK activity is regulated in a cell cycle-dependent manner and further showed that cells from scid/scid mice underwent permanent G 2 -M arrest after irradiation. In line with these results, inhibition of DNA-PK by DNA-PK-specific inhibitors also caused G 2 -M accumulation in response to irradiation and topoisomerase II poisons (46,47) and DNA-PKcs-deficient cells were found to accumulate in G 2 -M after IR (48).…”

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

“…The cell cycle dependency of NHEJ is even more contested, and in many reviews it is proposed to act only in G1 phase. This is due to the fact that a large majority of NHEJ-defective cells are hypersensitive to IR when irradiated in G1 but not in G2 (Stamato et al, 1988;Jeggo, 1990;Lee et al, 1997;Takata et al, 1998;Wang et al, 2001). Recently, this interpretation found molecular support by data showing that KU80 preferentially binds to DSBs in the G1 phase (Rodrigue et al, 2006).…”

Regulation and mechanisms of mammalian double-strand break repair

“…The relative contribution of each of these types of repair in mammalian DSB repair is controversial Haber, 2000;Johnson and Jasin, 2001), but it is generally believed that NHEJ plays a more important role than HRR in mitotically replicating cells (Jackson and Jeggo, 1995). HRR may play a more prominent role during meiosis and when sister chromatids are available during late S and G2 stages of the cell cycle (Haber, 2000;Morrison et al, 2000), whereas NHEJ is more important during G1 and early S stages (Lee SE et al, 1997;Takata et al, 1998).…”