Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells

Abstract

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70 Double-strand breaks (DSBs) are potentially catastrophic lesions that if not repaired will lead to loss of genetic information and mutagenesis or cell death. In mammalian cells, two major pathways exist to repair DSBshomologous recombination and nonhomologous endjoining (NHEJ;Liang et al. 1998). NHEJ, the rejoining of DNA ends with the use of little or no sequence homology, involves the processing of ends such that nucleotides are often deleted or inserted at the break site prior to ligation (Jeggo 1998). Such modifications are likely central to the ability of mammalian cells to rejoin DNA ends with a variety of structures. Homology-directed repair (HDR) of a DSB, in contrast, requires significant lengths of sequence homology so that a DNA end from one molecule can invade a homologous sequence and prime repair synthesis (Pâques and Haber 1999). Processing of DNA ends also occurs with HDR; however, repair is typically precise, because a homologous sequence templates the repair event.Several processes exist in which repair of a DSB is restricted to either NHEJ or HDR. For example, DSBs introduced by the RAG proteins to generate antigen receptor diversity during V(D)J rearrangement are repaired by the NHEJ pathway (Jeggo 1998), whereas those introduced during meiosis by the Spo11 protein are repaired by the HDR pathway (Keeney 2001). The restriction in type of DSB repair raises the question as to how pathway choice is regulated. Several studies point to cell cycle phase as one factor that modulates repair pathway choice. In chicken cells, HDR has been found to play a dominant role in repairing radiation-induced DSBs in late S/G 2 phase, whereas NHEJ is preferentially used during G 1 /early S phase (Takata et al. 1998). Consistent with this in mammalian cells, the preferred homologous template for HDR-the sister chromatid (Johnson and Jasin 2001)-is present only during the S/G 2 phase of the cell cycle. Despite a cell cycle preference, HDR and NHEJ can nevertheless be coupled for the repair of a single DSB in mammalian cells (Richardson and Jasin 2000), indicating that the two repair pathways are not completely restricted to different cell cycle phases and that other factors influence pathway choice.Based on in vitro studies of DNA end-binding proteins such as RAD52, it has been suggested that end-binding proteins may direct entry into alternative DSB repair pathways (Van Dyck et al. 1999). However, no evidence yet exists in vivo to support this model, and mutation of the Rad52 gene in mouse does not confer a cellular DSB repair phenotype (Rijkers et al. 1998). NHEJ proteins, that is, th...

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

“…This underlines the importance of coupling the regulation of the alternative DNA repair pathways and the control of the G1/S transition. Consistently, all the cell lines used in the different laboratories, showing over-stimulation of HR in NHEJdefective backgrounds (Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002), were defective in G1 arrest. They were either p53 À hamster cells or mouse embryonic stem cells, in which p53 is cytoplasmic and unable to control the G1 checkpoint (Aladjem et al, 1998).…”

“…NHEJ and HR indirectly compete through S phase for DSB repair, in the absence of G1 arrest Defects in NHEJ lead to a over-stimulation of HR specifically induced by DSBs (Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002). As HR does not act in G1, and NHEJ has been proposed to act throughout the cell cycle (Saintigny et al, 2001;Rothkamm et al, 2003;Saleh-Gohari and Helleday, 2004;Esashi et al, 2005), the simplest explanation was that the HR over-stimulation reflected the defect in NHEJ in S and G2 phases.…”

“…The two processes can compete or in contrast be coupled for DSB repair (Brouillette and Chartrand, 1987;Belmaaza et al, 1990;Belmaaza and Chartrand, 1994;Richardson and Jasin, 2000a;Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002). Consequently, the over-stimulation of HR in NHEJdefective cells (Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002) could easily be explained by defects in NHEJ in G2. To test this hypothesis, we measured IR-induced HR following irradiation in G1/ early S or late S/G2 of XRCC4 À or complemented cells.…”

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…We also evaluated the contribution of HP1 paralogs to the efficiency of HR using a previously characterized and widely used system to monitor homology-directed repair. 19,20 This system assesses the repair of a single DSB induced by the rare-cutting I-SceI endonuclease within a mutant GFP gene stably integrated in human cells, which, when repaired by homologous recombination, generates a wt-GFP. After 24-48 h, GFP expression can be detected by flow cytometry (Fig.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…It can be achieved either by mutating or silencing the genes coding for the proteins specifically involved in these pathways or by using chemicals. For example, a significant increase in HDR was obtained by silencing individually or simultaneously proteins involved in NHEJ (ligase IV, Ku70 and Ku80) in human and mouse cells [45,65,98,99]. In plants, the same type of results was obtained when using ZFNs targeting the endogenous ADH1 locus in Arabidopsis thaliana.…”