MRE11-RAD50-NBS1 complex dictates DNA repair independent of H2AX - PubMed (original) (raw)

MRE11-RAD50-NBS1 complex dictates DNA repair independent of H2AX

Jingsong Yuan et al. J Biol Chem. 2010.

Abstract

DNA double-strand breaks (DSBs) represent one of the most serious forms of DNA damage that can occur in the genome. Here, we show that the DSB-induced signaling cascade and homologous recombination (HR)-mediated DSB repair pathway can be genetically separated. We demonstrate that the MRE11-RAD50-NBS1 (MRN) complex acts to promote DNA end resection and the generation of single-stranded DNA, which is critically important for HR repair. These functions of the MRN complex can occur independently of the H2AX-mediated DNA damage signaling cascade, which promotes stable accumulation of other signaling and repair proteins such as 53BP1 and BRCA1 to sites of DNA damage. Nevertheless, mild defects in HR repair are observed in H2AX-deficient cells, suggesting that the H2AX-dependent DNA damage-signaling cascade assists DNA repair. We propose that the MRN complex is responsible for the initial recognition of DSBs and works together with both CtIP and the H2AX-dependent DNA damage-signaling cascade to facilitate repair by HR and regulate DNA damage checkpoints.

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Figures

FIGURE 1.

FIGURE 1.

The MRN complex is responsible for the initial recruitment of repair and signaling proteins to sites of DNA DSBs. A, 53BP1 transiently localizes to focus structure after IR treatment in H2AX−/− cells. H2AX+/+ and H2AX−/− were irradiated (3 Gy) and fixed at various time points after IR. The cells were immunostained with 53BP1 antibody (green). B, the initial 53BP1 focus formation observed in H2AX−/− cells depends on NBS1. H2AX+/+ and H2AX−/− were transfected with control siRNA or siRNA specifically targeting mouse NBS1 (simNBS1). 72 h later, the cells were irradiated (3 Gy) and fixed at 30 min or 4 h after IR. Immunostaining was carried out with anti-γH2AX, anti-53BP1, or anti-mouse NBS1 antibodies. C, quantification of 53BP1 foci following DNA damage. The data are presented as percentages of cells containing 53BP1 foci. The means and standard deviation (error bars) shown are obtained from three independent experiments in which more than 200 cells were counted. D, the initial focus localization of 53BP1 and BRCA1 requires the MRN complex. Double depletion of H2AX and NBS1 or of H2AX and MRE11 was performed in U2OS cells (see “Experimental Procedures” for details). Following siRNA transfection, the cells were irradiated (5 Gy) and fixed 30 min later. Immunostaining was carried out using the indicated antibodies. E, quantification of 53BP1 and BRCA1 focus formation following DNA damage. The data presented are percentages of cells containing 53BP1 or BRCA1 foci 30 min after DNA damage. The means and standard deviation (error bars) shown are results obtained from three independent experiments in which more than 200 cells were counted. Bars, 10 μm.

FIGURE 2.

FIGURE 2.

The transient recruitment of 53BP1 depends on NBS1 but not MDC1 or RNF8. A, transient recruitment of 53BP1 upon DNA damage still occur in RNF8−/− and MDC1−/− MEFs. B, the transient recruitment of 53BP1 upon DNA damage was abolished by NBS1 depletion in RNF8−/− and MDC1−/− MEFs. The cells with or without siRNA transfection were irradiated and fixed at indicated time points after IR. Immunostaining was carried out with the indicated antibodies. Bars, 10 μm.

FIGURE 3.

FIGURE 3.

MRN complex and CtIP, but not H2AX, are required for efficient HR repair. A and B, depletion of components of the MRN complex or CtIP impairs RPA focus formation (A) but does not affect focus formation of other DNA damage repair factors (B). U2OS cells were transfected with indicated siRNAs. 48 h later, the cells were irradiated (10 Gy) and allowed to recover for 6 h before fixation and immunostaining with antibodies as indicated. C and D, siRNA-mediated down-regulation of various DNA damage and repair proteins were carried out in U2OS DR-GFP cells. The knockdown efficiency using indicated siRNAs was confirmed by immunoblotting (C). The percentage of GFP positive cells was determined by flow cytometry 48 h after cells were electroporated with pCBASce plasmid (D). The means and standard deviation (error bars) shown are obtained from three independent experiments. Asterisk in C, nonspecific band. Bars, 10 μm. DAPI, 4′,6′-diamino-2-phenylindole.

FIGURE 4.

FIGURE 4.

IR-induced RPA focus formation is largely independent of ATM, γH2AX, and MDC1. A and B, IR-induced RPA focus formation was observed in ATM-, H2AX-, or MDC1-deficient cells. The cells with the indicated genotypes were irradiated (10 Gy) and allowed to recover for 6 h before fixation and immunostaining. C, inhibiting ATM activity by ATM inhibitor KU55933 did not impair RPA focus formation following DNA damage. U2OS cells were pretreated with dimethyl sulfoxide (DMSO) or KU55933 at the indicated concentrations for 1 h before they were exposed to IR (10 Gy). Immunostaining was performed 6 h after IR using anti-RPA2 and ATM pS1981 antibodies. Bars, 10 μm.

FIGURE 5.

FIGURE 5.

A proposed revised model of mammalian DNA damage response. We hypothesize that there are two separate pathways involved in the recruitment and accumulation of DNA damage checkpoint and repair proteins at DSBs. One of them is the H2AX-independent pathway, which requires the MRN complex and probably limits the localization of these checkpoint and repair proteins at or closer to sites of DNA breaks. We speculate that this pathway is mainly geared toward DNA repair but also initiates ATM activation and cell cycle checkpoint. The second pathway is the well studied H2AX-dependent pathway, which allows the accumulation of DNA repair proteins and the spreading of DNA damage signaling proteins to larger chromatin regions surrounding the damaged sites. This H2AX-dependent pathway carries out at least two functions: one is to facilitate DNA repair, and the other is to promote the amplification of DNA damage signals, which lead to a full checkpoint activation and allow time for the completion of DNA repair.

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