Characterization of homologous recombination induced by replication inhibition in mammalian cells

Abstract

To analyze relationships between replication and homologous recombination in mammalian cells, we used replication inhibitors to treat mouse and hamster cell lines containing tandem repeat recombination substrates. In the first step, few double-strand breaks (DSBs) are produced, recombination is slightly increased, but cell lines defective in non-homologous end-joining (NHEJ) affected in ku86 (xrs6) or xrcc4 (XR-1) genes show enhanced sensitivity to replication inhibitors. In the second step, replication inhibition leads to coordinated kinetics of DSB accumulation, Rad51 foci formation and RAD51-dependent gene conversion stimulation. In xrs6 as well as XR-1 cell lines, Rad51 foci accumulate more rapidly compared with their respective controls. We propose that replication inhibition produces DSBs, which are first processed by the NHEJ; then, following DSB accumulation, RAD51 recombination can act.

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

“…We also show that the increase in IR-induced HR observed in XRCC4 À cells corresponds to cells irradiated in G1, but not in late S/G2 phase (Figure 1e). Noticeably, XRCC4 À cells were sensitive to IR in mid-S phase and showed an intermediate level of HR over-stimulation, suggesting an involvement of NHEJ during S phase, as already proposed (Saintigny et al, 2001;Mills et al, 2004). These results were further confirmed with a third protocol, using mimosine to block cells at late G1/early S (see below, Figure 3).…”

“…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. Surprisingly, we show here that, (1) an NHEJ defect in G1, but not in G2, stimulated IR-induced HR; (2) the cell cycle restriction for HR, that is in S/G2 phase, was not affected by the NHEJ defect; (3) the absence of delay at the G1/S transition was essential for the overstimulation of IR-induced HR in XRCC4 À cells; this allowed the presence in S/G2 of unrepaired DSB generated in G1.…”

“…Recently, this interpretation found molecular support by data showing that KU80 preferentially binds to DSBs in the G1 phase (Rodrigue et al, 2006). However, this view is challenged by the fact that NHEJ has been proposed to act in S phase (Saintigny et al, 2001;Mills et al, 2004) and throughout the cell cycle (Rothkamm et al, 2003). Supporting the latter view, crosstalk between HR and NHEJ has been described, suggesting overlaps during the cell cycle.…”

“…DNA double-strand breaks (DSBs), which can be produced by ionizing radiation (IR) or replication inhibition (Jeggo et al, 1995;Rothstein et al, 2000;Saintigny et al, 2001), are highly toxic lesions which can lead to profound genome rearrangements. On the other hand, DSBs can also be advantageously used to generate genetic diversity in physiological processes such as meiosis or V(D)J recombination (Kleckner, 1996;Smith and Jackson, 1999;Cohen and Pollard, 2001;Jung and Alt, 2004).…”

SIRT 7 promotes genome integrity and modulates non‐homologous end joining DNA repair

“…In addition, our results indicate that SIRT7 deficiency leads to replication stress (Figs 4G–I and EV2A–C). Replication stress might be a consequence of impaired NHEJ, as has been reported previously (Saintigny et al , 2001; Lundin et al , 2002). However, we cannot rule out that SIRT7 might have an effect on chromatin structure at the replication fork, which has been suggested for other histone deacetylases (Bhaskara et al , 2013; Wells et al , 2013), and/or by directly targeting the DNA replication machinery.…”