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Human Molecular Genetics Advance Access originally published online on August 8, 2005
Human Molecular Genetics 2005 14(18):2685-2693; doi:10.1093/hmg/ddi302
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© The Author 2005. Published by Oxford University Press. All rights reserved.
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Rad50 depletion impacts upon ATR-dependent DNA damage responses

Hui Zhong, Alyson Bryson, Mark Eckersdorff and David O. Ferguson*

Department of Pathology, University of Michigan, Ann Arbor, MI 48109-0602, USA

* To whom correspondence should be addressed. Tel: +1 7347644591; Fax: +1 7346153441; Email: daviferg{at}umich.edu

Received May 30, 2005; Accepted August 2, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, we tested whether MRN has a global controlling role over the ATR kinase through the study of MRN deficiencies generated via RNA interference. We show for the first time that MRN is required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. We have uncovered novel phenotypes caused by MRN deficiency that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to our understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The complex comprising Mre11, Rad50 and NBS1 (collectively termed MRN) is a central component in the cellular response to ionizing radiation (IR) and other causes of DNA double-strand breaks (DSBs) (1Go). Partial loss of function mutations in the NBS1 and Mre11 genes lead to the genomic instability disorders Nijmegen breakage syndrome (NBS) and ataxia telangiectasia like disorder (ATLD), respectively (2Go–4Go). Both syndromes feature cellular and whole body hypersensitivity to IR as well as neurological defects. NBS leads to strong cancer predisposition, developmental delay and mental retardation, whereas the clinical features of ATLD are milder.

The MRN complex has dual roles in the response to DSBs; repair of the breaks themselves as well as control of cell cycle checkpoints triggered by the breaks (5Go). Although the precise roles of MRN at DSBs are not yet known, the complex possesses several biochemical activities and structural features related to direct involvement in repair. Mre11 possesses 3' to 5' double-stranded DNA exonuclease activity and single- and double-stranded endonuclease activities (5Go). Rad50 is an ATPase with regional homology to the structural maintenance of chromosomes family involved in multiple aspects of chromosome dynamics (6Go). Structural and biochemical studies indicate that Mre11–Rad50 heterodimers possess DNA binding, nuclease and ATPase activities at one end of a long coiled coil and a large hook on the opposite end. Higher order interlinking of these structures may maintain DNA breaks in close proximity during nucleolytic processing of termini (7Go).

MRN influences cell cycle checkpoints triggered by DSBs through control of the ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Mutations in the ATM gene are responsible for the syndrome ataxia telangiectasia (A-T) which features IR hypersensitivity and cancer predisposition (8Go). The role of ATM is facilitated through DNA damage induction of its kinase activity (9Go) and relocation to sites of damage (10Go). It is now established that the presence of the MRN complex is required for ATM-dependent phosphorylation of several critical cell cycle checkpoint factors including Chk1, Chk2 and p53 (11Go–15Go).

Although ATM is the master controller of the DSB response, the A-T and Rad3 related (ATR) kinase controls many responses to replication stress which are experimentally induced by low-dose ultraviolet (UV) radiation or chemical inhibitors such as hydroxyurea (16Go–18Go). ATM and ATR are related through regional sequence homology and are members of the phosphoinositol 3-kinase related kinase (PIKK) family of serine–threonine protein kinases (8Go). The dichotomy of function between ATM and ATR is best illustrated by phenotypic comparisons of mammalian cells with mutant alleles of ATM which are hypersensitive to IR, but not to UV, whereas patient cells from the recently described Seckel-ATR syndrome which express very low levels of ATR display the reverse (8Go,16Go).

ATM and ATR each phosphorylate numerous substrates after different forms of genotoxic stress. These substrates have a wide range of functions, including protein kinases such as Chk1 and Chk2 which further propagate damage signals, DNA repair factors such as Rad17, Mre11 and NBS1, and proteins that act within chromatin such as H2AX and structural maintenance of chromosomes 1 (Smc1) (19Go). Control over functions of this wide range of substrates ultimately dictates biological responses to genotoxic stresses. These responses include cell cycle checkpoints which either cause cells to cease dividing to allow time for repair or induce apoptosis to eliminate dysfunctional cells. ATM plays a role in all three major DSB induced checkpoints; within S phase and at the G1/S and G2/M transitions (8Go). Upon exposure to low-dose UV, ATR plays a role at checkpoints within S phase and at the G2/M transition (17Go,20Go). In addition, other types of damage responses exist, such as UV induced degradation of the cyclin-dependent kinase inhibitor (CDKI) p21 (21Go,22Go). Although not known to be a direct substrate, the p21 degradation response is dependent on the ATR kinase.

ATM and ATR play critical roles in ensuring DNA is properly repaired and in protecting the genome from instability; therefore, understanding how these kinases themselves are regulated is of fundamental importance. While it is well established that MRN controls certain functions of ATM, comparison of human syndromes and experimental mouse models suggests that this complex has significant functions outside of this role. For example, NBS patients are more affected at birth than A-T patients and mice tolerate engineered ATM null alleles, whereas nullizygosity for MRN components confers early embryonic lethality (1Go). Hypothetically, the relative severity of MRN deficiencies could be explained by control over ATM-independent processes such as those governed by ATR. However, it was recently shown that a cell line harboring a partial loss of function Mre11 mutation and reduced levels of full-length NBS1 and Rad50 maintains normal ATR function (17Go). In this same study, cell lines containing the common NBS1 partial loss of function allele did show reduced ATR-dependent phosphorylation toward a subset of substrates. This subset included the Chk1 kinase and p53, but not H2AX, leading to the hypothesis that a defect was present, but only involving signaling molecules and not factors associated with DNA or within chromatin near sites of damage. Whether these observations reflect a true functional relationship among these factors, or result indirectly from complex effects of disease-associated hypomorphic (or gain of function) alleles is not known.

We have directly tested whether a functional relationship exists between the MRN complex and ATR and have done so by generating MRN deficiencies which avoid the complexities of hypomorphic disease-associated alleles. The work herein demonstrates that MRN is indeed required for several ATR functions. We have discovered that these functions include phosphorylation of Smc1, indicating that the control of ATR by MRN is not restricted to signaling factors. In addition, we have uncovered previously unknown phenotypes caused by MRN deficiency that strongly support the notion of a functional link between this complex, ATR and Smc1. Although MRN exerts broad control over ATR, including phosphorylation of both signaling and chromatin-associated factors, we have also found that some ATR-dependent responses do not require MRN. These findings have significant implications regarding mechanisms of communication between DNA damage detectors and effectors of cellular responses, which cooperate to maintain genomic stability and prevent neoplastic transformation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA interference of human Rad50
To investigate the roles of the MRN complex beyond ATM-dependent DSB repair, we generated deficiencies of full-length proteins employing a combination of stable RNAi using short hairpin RNAs (shRNA) and transient RNAi using standard double-stranded RNA oligonucleotides. To potentially maximize the impact on MRN, we targeted Rad50 because it is a large structural protein central to the conformation and function of the complex (7Go).

We generated stably expressing hairpin RNAi clones in the HCT116 line, which was chosen because it possesses a stable diploid karyotype necessary for chromosome stability studies (23Go). In addition, although readily detectable, the MRN component proteins are less abundant in HCT116 relative to many other lines (24Go); therefore we reasoned that RNAi is likely to be highly effective in reducing protein levels to critically low concentrations. Stable Rad50 knockdown clones were generated through the integration of a neomycin selectable plasmid containing a 19 bp inverted sequence matching a region of human Rad50 mRNA, flanking a seven base spacer (see Materials and Methods). Expression driven by the U6 promoter generates a double-stranded RNA in a hairpin configuration, a structure known to cause effective RNA interference in mammalian cells (25Go,26Go). Western blot analyses indicated that Rad50 protein was reduced in 24 clones (data not shown). Two clones with the greatest stable reduction of Rad50 were expanded for experimentation (Fig. 1A). Two neomycin resistant clones generated through the integration of an ‘empty vector’ were used as controls. We also succeeded in generating additional stable Rad50 deficient and control lines using 293T cells (Fig. 1A). Transient transfection of double-stranded RNAi oligonucleotides (27Go) was performed in 293T cells and produced severe reduction of Rad50 by 48 hours (Fig. 1A), the time at which experiments using this approach were performed.



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  		Figure 1. RNA interference of Rad50 recapitulates hallmarks of MRN deficiency. (A) Stable suppression of Rad50 levels by short hairpin RNAi in the HCT116 and 293T cell lines and transient suppression in 293T (48 h after transfection). Immunoblotting was performed with antibodies to the indicated proteins. R4, R5, R10, independent stable MRN deficient lines; Ctr, control line stably transfected with empty vector; siRad50, transient knockdown of Rad50; Tubulin, loading control. (B) RNAi induced MRN deficiency confers IR hypersensitivity. The Y-axis indicates the percent of cells surviving relative to unirradiated control populations and the X-axis indicates increasing doses. Error bars represent standard deviation of three independent experiments. R4 and R5, independent stable MRN deficient lines; C1 and C2, independent control lines. (C) RNAi induced MRN deficiency confers a defective IR responsive intra-S phase checkpoint. Radioresistant DNA synthesis was assessed in 293T cells 1 h after the indicated dose of {gamma} radiation applied 48 h after transfection with Rad50 siRNA or mock. The Y-axis represents the relative levels of DNA synthesis at the indicated doses of {gamma} radiation when compared with unirradiated controls. Error bars indicate standard deviation of three independent experiments.

 
Using both stable and transient RNAi, we find that deficiency of Rad50 is associated with reduced levels of Mre11 and NBS1 (termed MRN deficient) (Fig. 1A). This suggests that Rad50 is required for protein stability of the other members and allowed us to examine effects of deficiency of the entire complex. The stable MRN deficient HCT116 lines pursued for study remained viable and stably deficient for at least 5 months (Fig. 1A), although they and the stable 293T lines proliferate somewhat slower compared with controls (data not shown).

RNA interference of Rad50 recapitulates hallmarks of MRN deficiencies
To establish the validity of the RNAi approach, we determined whether our Rad50 knockdowns faithfully recapitulate established hallmarks of MRN deficiencies. Previous studies of cells from NBS and ATLD patients and experimental models have established that MRN is required for maintaining chromosomal stability, resistance to IR exposure, IR induced cell cycle checkpoints and other roles (1Go). We find that stable reduction in HCT116 confers hypersensitivity to IR (Fig. 1B) and increased spontaneous chromosomal instability (Table 1). In 293T cells, we find that transient MRN deficiency causes a defective intra-S phase checkpoint after IR as reflected by continued nucleotide incorporation, which is significantly reduced in control cells (Fig. 1C). Therefore, regarding the survival after IR exposure, the IR induced intra-S phase checkpoint and maintenance of chromosomal stability, the RNAi approach employed here recapitulates prior findings and establishes it as a viable strategy to discover and delineate roles of the MRN complex.


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  		Table 1. Spontaneous and UV induced genomic instability is enhanced in MRN deficiency
 
MRN complex controls ATR-dependent phosphorylation of Smc1 and Chk1
Smc1 is a chromosomal protein member of the cohesin complex involved in sister chromatid cohesion in the wake of DNA replication and has been implicated in DNA repair via homologous recombination (6Go). Phosphorylation on serines 957 and 966 is required for radioresistance and the intra-S phase checkpoint and is ATM dependent after IR and ATR dependent following low-dose UV exposure (10Go,14Go,20Go). In the case of IR exposure, phosphorylation is also dependent on NBS1 and full-length Mre11 (10Go,28Go). However, the relationship between Smc1 and the MRN complex in the context of ATR function has not been explored.

Therefore, we examined phosphorylation at serine 957 in Smc1 in acute time intervals after low-dose UV exposure. In both the stable HCT116 and the transient 293T MRN deficiencies, an obvious reduction in phosphorylation is seen (Fig. 2A). Levels of total Smc1 protein were not affected by MRN deficiency nor were they altered by UV exposure. We wished to confirm that the phosphorylation of serine 957 of Smc1 at the 5 J/m2 dose of UV employed is ATR dependent. To accomplish this, we utilized a lymphoblastoid cell line derived from a Seckel-ATR patient, in which very low levels of full-length ATR are present due to a mutation effecting splicing of the transcript (16Go). When compared with a line from an unaffected sibling, significantly lower levels of UV induced phosphorylation of Smc1 were observed in Seckel-ATR, confirming that this modification is ATR dependent (Fig. 2B). Residual signal detected in Seckel-ATR cells likely results from the residual ATR present in this syndrome. In addition, there was no apparent effect of ATM deficiency (Fig. 2B).



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  		Figure 2. MRN controls ATR-dependent phosphorylation of Smc1 and Chk1. (A) UV induced phosphorylation of Smc1 and Chk1. Immunoblotting was performed on whole cell lysates with the indicated antibodies at the designated time intervals after 5 J/m2 UV irradiation. (–), no UV exposure; Tubulin, loading control. Each blot is a representative example of at least three independent experiments. (Left panel) R5 MRN deficient HCT116 line (R5) compared with controls (Ctrl). (Right panel) 293T cells with transient reduction of MRN (+) compared with mock transfection (–), 48 h after transfection. Numbers below certain western blots indicate relative levels determined by software-based quantitation of the representative experiment shown. (B) ATM or ATR dependence of UV induced phosphorylation. Immunoblotting and quantitation performed as in (A). s=Seckel-ATR allele.

 
We also examined UV induced phosphorylation of the Chk1 kinase. Chk1 is phosphorylated and activated by ATM in response to IR (29Go,30Go) and by ATR in response to UV or other causes of stalled replication (17Go,18Go,30Go–33Go). We find that UV induced phosphorylation of Chk1 is reduced in MRN deficiency (Fig. 2A). Chk1 maintains a baseline level of activity in the absence of exogenously induced DNA damage (34Go). This baseline level of activity is likely reflected as detectable phosphorylated Chk1 in undamaged cells, as has been observed to varying degrees in different mammalian cell lines (11Go,17Go,30Go). The baseline phosphorylated Chk1 is particularly prevalent in HCT116, making the induction difficult to discern visually. Therefore, we quantitated band intensities from three independent experiments. The control was induced an average of 1.89- and 1.77-fold at 0.5 and 1.5 h after UV, respectively, whereas the R5 line was induced 1.27- and 1.07-fold. The differences in induction between the lines were statistically significant (P<0.01). The differences in Chk1 phosphorylation in the 293T transient transfection and in the Seckel-ATR line (discussed subsequently) were more obvious. Interestingly, these two components may reflect distinct roles of activated Chk1, and only the damage-induced component is influenced by the MRN complex. We have confirmed that under our experimental conditions UV induced phosphorylation of Chk1 is reduced in ATR deficiency and is independent of ATM (Fig. 2B) (33Go). We were unable to find detectable levels of spontaneous phosphorylated Chk1 in the A-T and Seckel lines, so we cannot comment on which kinase (if either) may be responsible for the uninduced component. Nonetheless, we have demonstrated that the MRN complex is required for ATR-dependent damage-induced phosphorylation of Smc1 and Chk1.

Phenotypes of MRN deficiency support a link to ATR functions
If the MRN complex plays biologically significant roles which involve ATR, we surmised that our MRN deficient cells would display phenotypes similar to those previously established to be caused by ATR deficiency. Thus, we exposed two stable MRN deficient lines (R4 and R5) to increasing UV doses. Indeed, both knockdown lines tested are hypersensitive to UV relative to control lines stably transfected with empty vector (Fig. 3A). The degree of hypersensitivity and overall kinetics of the curves resemble those associated with ATR deficiency, which is less severe than hypersensitivity conferred by defective nucleotide excision repair (NER) (16Go). Deficiency of other factors implicated in the response to replication blockage confer similar degrees of mild UV hypersensitivity (35Go,36Go), which might reflect differences in function between these factors and the highly specific NER components. It is also possible that the degree of hypersensitivity we observe for MRN deficiency underestimates the importance of this complex in survival after UV exposure due to residual protein present during RNA interference.



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  		Figure 3. Phenotypes of MRN deficiency support a link to ATR functions. (A) Stable MRN deficiency confers UV hypersensitivity. The Y-axis indicates the percent of cells surviving relative to unirradiated control populations and the X-axis indicates increasing doses. Error bars represent standard deviation of three independent experiments. R4 and R5, independent MRN deficient lines; C1 and C2, independent control lines. (B) Defective UV induced intra-S checkpoint in stable MRN deficient cells. Radioresistant DNA synthesis was assessed in MRN deficient (R10) and control 293T cell lines 1 h after the indicated doses of UV radiation. Error bars indicate standard deviation of three independent experiments. (C) Defective UV responsive G2/M checkpoint in MRN deficient cells. Exponentially growing cultures of HCT116 transfected with empty vector (white bars) and MRN deficient R-5 line (black bars) were harvested at the indicated times after 5 J/m2 of UV radiation. Untreated cultures were designated T=0. The percentage of mitotic cells (based on phospho-H3 staining) is plotted. Error bars indicate standard deviation of three independent experiments. (D) (Top) Representative examples of UV induced chromatid gaps (arrows) in the MRN deficient HCT116 R5 cell line (DAPI stain). (Bottom) Two UV induced translocations (arrows) in the MRN deficient R5 HCT116 cell line (DAPI—left and SKY—right).

 
We next examined the integrity of the intra-S and G2/M checkpoints after UV exposure. The intra-S phase checkpoint, as measured by damage-induced reduction of nucleotide incorporation, is dependent on ATR and independent of ATM after low doses of UV (20Go). We find that 293T cells rendered stably deficient in MRN exhibit a significant defect in this checkpoint (Fig. 3B). The G2/M checkpoint after low-dose UV prevents cells from entering mitosis with damaged or unreplicated regions of DNA and was recently shown to be deficient in cells from Seckel-ATR (37Go) and NBS patients (17Go). Through the detection of the phosphorylated form of histone H3, a commonly used marker for entry into mitosis (38Go), we observe that a significantly higher percentage of cells escape G2 and enter mitosis 3 h after a UV dose of 5 J/m2 when rendered stably MRN deficient (Fig. 3C). Therefore, after UV exposure, the MRN complex is required for survival and fully functional intra-S and G2/M cell cycle checkpoints, which have been shown by others to require the ATR kinase (16Go,17Go,20Go,37Go).

MRN deficiency enhances UV induced genomic instability
To gain further insight into the related roles of MRN and ATR, we determined the cytogenetic consequences of UV exposure in MRN deficiency and controls using two methods: (i) standard DAPI staining to quantify chromosomal anomalies such as breaks and gaps which reflect lack of repair and (ii) spectral karyotyping (SKY) to reveal translocations which indicate erroneous repair (39Go). We find that UV radiation enhanced chromosomal instability in both control and stable MRN deficiency when examining metaphases 24 h after exposure (Fig. 3D and Table 1). By 48 h after exposure, the frequency of chromosomal anomalies per metaphase in control cells had decreased (0.33 to 0.17). In contrast, the frequency in MRN deficient cells did not decrease at 48 h after UV (0.98 to 0.99), indicating that lesions persist in the absence of this complex. SKY analysis revealed that UV induced translocations are more frequent in MRN deficiency, suggesting that persistent UV induced lesions can undergo errant DNA repair. In both untreated and UV exposed MRN deficient cells, frequent gaps in single chromatid were observed. Importantly, these gaps represent segments of missing chromatid, consistent with MRN functioning to ensure complete replication through spontaneous or UV induced lesions (40Go).

MRN complex is not required for all ATR-dependent responses
A unique example of an ATR-dependent response involves the p21 CDKI. After IR, p21 controls the G1/S cell cycle checkpoint through its CDKI activity and through direct interaction and inhibition of the DNA replication processivity factor, PCNA (21Go). In contrast, after low-dose UV radiation (<40 J/m2), p21 does not influence the G1/S checkpoint and instead undergoes ubiquitin mediated protein degradation, which is dependent on ATR (22Go).

We wished to determine whether MRN has influence over this response. In both control and MRN deficiencies, there is a dramatic decrease in p21 protein levels 1.5 h after a UV dose of 5 J/m2 (Fig. 4A). However, without UV exposure, the p21 signal is stronger in MRN deficiency compared with controls, making it difficult to visually compare the degradation responses. Therefore, we quantitated the bands and calculated the relative p21 levels after UV compared with the unexposed level for each line (Fig. 4A). These values indicate that MRN deficiency does not negatively impact the UV induced p21 degradation response. We confirmed that this response is ATR dependent by examining p21 levels in Seckel-ATR cells after UV exposure. Similar to the previously reported effect of conditional inactivation of ATR (22Go), the Seckel-ATR cells show less reduction in p21 levels by 1.5 h after UV (Fig. 4B). The ATR deficient cells did show a slight decrease in p21, again likely due to the residual full-length protein present in this syndrome. To determine whether the difference in reduced p21 levels is significant we quantitated western blot signals from three independent experiments. At 1.5 h after UV, p21 levels dropped to 27% of unirradiated in control compared with 64% in Seckel-ATR, a difference that was statistically significant (P<0.01) (Fig. 4B). Thus, we have confirmed that ATR governs the UV induced p21 degradation response and have shown that it is not impacted upon by reduced MRN levels.



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  		Figure 4. ATR-dependent responses not effected by MRN deficiency. (A) UV induced p21 degradation and H2AX phosphorylation. Immunoblotting was performed on whole cell lysates with the indicated antibodies at the designated time intervals after 5 J/m2 UV irradiation. (–) indicates no UV exposure. Tubulin, loading control. Each blot is a representative example of at least three independent experiments. (Left panel) The R5 MRN deficient HCT116 line (R5) compared with control (Ctrl). (Right panel) 293T cells with transient reduction of MRN (+) compared with mock transfection (–), 48 h after transfection. Numbers below certain western blots indicate relative levels determined by software-based quantitation in the representative experiment shown. (B) p21 degradation and H2AX phosphorylation are ATR dependent after 5 J/m2 UV exposure. Immunoblotting and quantitation performed as in (A). s=Seckel-ATR allele.

 
H2AX is a core histone variant distributed throughout chromosomes and is required for genomic stability and cellular survival after DNA damage (41Go,42Go). It is phosphorylated on its C-terminus (referred to as {gamma}-H2AX) at sites of DNA damage and surrounding megabase regions (43Go). Like Smc1, phosphorylation of H2AX is dependent on ATM after IR (44Go) and on ATR during replication stress induced by UV or hydroxyurea (16Go,45Go). We measured UV induced formation of {gamma}-H2AX to determine whether the MRN complex is required for this response. {gamma}-H2AX was increased by 0.5 h after UV in both MRN deficient and control cells (Fig. 4A). Interestingly, at each time point after UV, {gamma}-H2AX signal is higher in MRN deficiency than that in controls. We confirmed that the UV induced phosphorylation is unaffected by ATM deficiency and is significantly reduced in Seckel-ATR (P<0.01 at 0.5 and 1.5 h after UV) (Fig. 4B). Therefore, unlike Smc1 and Chk1, ATR-dependent phosphorylation of H2AX is not negatively impacted by transient or stable reduction of MRN levels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we have shown for the first time that the MRN complex is required for ATR-dependent phosphorylation of a chromatin-associated factor (Smc1) and that the MRN complex is required for cellular survival, an intra-S phase checkpoint, and genomic stability after low-dose UV radiation. In addition, we have shown that ATR-dependent phosphorylation of Chk1 and the UV induced G2/M checkpoint do indeed depend on the MRN complex and are not phenotypes restricted to patient cells harboring the common NBS1675{Delta}5 partial loss of function allele. Thus, this work extends our understanding of the crucial role that the MRN plays over a broad range of DNA damage responses.

The evidence now indicates that MRN has influence over both ATR and ATM after diverse genotoxic stresses. Our findings have strong implications in this regard. For example, given the disparate roles of Smc1 and Chk1, ATR-dependent phosphorylation influenced by MRN cannot easily be categorized, such as function at damage sites versus diffuse signaling. Furthermore, we find that certain ATR-dependent responses may not require MRN. UV induced H2AX phosphorylation occurs robustly in MRN deficiency, consistent with the observation that it can occur efficiently in patient cells with the common NBS1675{Delta}5 hypomorphic allele (17Go). The p21 CDKI plays complex roles after DNA damage. Although much remains to be learned about the purpose and mechanisms of the UV induced p21 degradation response, we have shown that it occurs in MRN deficiency and is reduced in Seckel-ATR syndrome. Together, these data imply that MRN is not universally required for functions of the ATR kinase, rather it controls a subset of responses. However, it is also possible that another kinase such as DNA-PK can substitute for ATR upon selected substrates (46Go) or that certain substrates may remain efficiently phosphorylated in the presence of residual amounts of the MRN complex.

We have found that MRN deficiency enhances UV induced genomic instability with a particular tendency to cause single chromatid gaps. UV irradiation generates helix distorting pyrimidine dimers which can be repaired by the NER pathway or bypassed by specialized DNA polymerases (47Go). However, unrepaired lesions can block replication fork movement. Consistent with the notion that MRN plays roles at sites of blocked replication, UV irradiation of cells lacking the bypass polymerase eta causes the MRN complex to form increased number of foci during S phase (48Go). The chromatid gaps that we observe may therefore result from the absence of MRN functions in S phase that would normally occur within these foci. The persistence of these gaps into M phase is consistent with deficiency of the MRN dependent G2/M checkpoint. Indeed, it has also been noted that frequent chromatid gaps are present in cells rendered deficient in ATR (18Go,23Go) or Smc1 (49Go) further indicating a functional relationship among these factors in which they serve to prevent propagation of detrimental chromosomal aberrations.

There are several mechanisms by which MRN has been proposed to control ATM after DNA DSBs. These include control of activating ATM autophosphorylation (9Go), indirect effects in which the Mre11 nuclease initiates a signal through the generation of single-stranded DNA (5Go) and control of ATM localization to sites of DSBs (10Go). Recently, significant advances have been made in our understanding of the interplay between MRN and ATM at sites of DSBs through analyses of these factors in vitro (50Go). A tetramer of two Mre11 and two Rad50 molecules was found to bind to DNA ends, whereas NBS acted as an adapter to provide direct association with catalytically inactive ATM dimers. Monomerization and activation of ATM did not occur until single-stranded DNA was generated through duplex unwinding activity provided by the Rad50 ATPase. ATM was proposed to subsequently phosphorylate local substrates and then diffuse away from MRN to act more distantly.

The adapter function of NBS1 is now known to represent a conserved strategy for recruiting PIKK kinases to sites of DNA damage (46Go). NBS1, ATR interacting protein (ATRIP) and Ku80 are required for localizing ATM, ATR and DNA-PKcs, respectively, to lesions through a conserved C-terminal domain in each of the adapters. Taken together, these and other studies suggest a model in which the crucial activating signal for ATM and ATR is the generation of single-stranded DNA at lesions that are initially double-stranded or in complex configurations that could arise, for example, at disrupted replication forks (51Go). Although direct interaction between NBS1 and ATM provides an explanation for the dependence of ATM activity on MRN, it raises the important question as to what links ATR to MRN. In the recent study by Stiff et al. (17Go), patient cells harboring the NBS1675{Delta}5 mutation were found deficient in the formation of damage-induced ATR foci. Our findings and those by Stiff et al. demonstrate the breadth of ATR-dependent phosphorylation events and cellular DNA damage responses under the influence of MRN, and together raise the specter that NBS1 could recruit ATR-ATRIP via a mechanism similar to the recruitment of ATM. The exact mechanism, however, could involve DNA unwinding or nucleolytic digestion by MRN without direct protein interaction or employ an undefined adapter protein which links MRN to ATR-ATRIP.

The possible mechanisms of the ATR-MRN link are not mutually exclusive and combinations of mechanisms may operate. Indeed, our findings that MRN is required for ATR-dependent phosphorylation of Smc1 but not H2AX and is required for UV induced cell cycle checkpoints but not p21 degradation suggest that the link between ATR and MRN does not reside with one simple adapter interaction, but is likely to be quite intricate. Therefore, the findings presented here represent a fundamental advance in our knowledge of the roles of the MRN complex in DNA damage detection which will undoubtedly stimulate numerous studies aimed at deciphering the exact nature of the newly uncovered ATR-MRN link.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
HCT116 cells and 293T cells from ATCC were cultured in McCOY'S 5A or DMEM medium, respectively, each supplemented with fetal bovine serum and antibiotics (Fisher). A-T (GM01588) and control SV40-transformed fibroblasts were kindly provided by Dr Mats Ljungman (University of Michigan) (52Go). ATR-Seckel (DK0061) and unaffected sibling (DK0063) lymphoblast lines were kindly provided by Drs Penny Jeggo and Mark O'Driscoll (16Go).

RNA interference
Coding sequences for RNAi were selected as described (25Go). Rad50 RNAi sequences correspond to NCBI accession NM-005732 (nucleotides 1921–1939), and NCBI-BLAST verified no significant homology to other genes. Short hairpin RNAi was generated using pSuppressorNeo vector (IMGENEX inc). 1x106 cells grown on 10 cm dishes were transfected with 6 µg DNA using Fugene6 (Roche). Transfected colonies were selected in 2 µg/ml G418 (Fisher) applied 3 days after the transfection and maintained thereafter. siRNA oligos (Invitrogen) directed at the same nucleotides were transfected at 200 µM on 6 cm dishes using 10 µl lipofectamine2000 (Invitrogen) according to the manufacturer's instructions.

Immunoblotting
Whole cell extracts from cells treated with 5 J/m2 in a FB-UVXL-1000 UV crosslinker (Fisher) or untreated were generated in ice-cold lysis buffer containing 25 mM HEPES (pH 7.4), 150 mM KCl, 10 mM MgCl2, 10% Glycerol, 2 mM DTT and protease inhibitor mixture (Roche). Lysates on ice were sonicated four times for 30 s and centrifuged at 13 000g for 60 min. Supernatant protein concentrations were determined by the Bradford assay (BioRad). Proteins were resolved on 7–15% SDS–PAGE and transferred onto nitrocellulose membranes. Primary antibodies were as follows: polyclonal {alpha}Rad50, {alpha}Mre11, {alpha}Nbs1 (Novus Biologicals), monoclonal {alpha}phospho-Chk1 (Ser 345), {alpha}phospho-Smc1 (Ser 957), {alpha}p21 (Cell Signaling Technology), {alpha}phospho-H2AX (Upstate Biotechnology), {alpha}Chk1 and {alpha}Smc1 (Santa Cruz). Horseradish peroxidase labeled secondary antibodies was from Jackson Labs. When necessary, figures were cropped using Adobe Photoshop software (Adobe inc). Quantitation of western blot bands was performed using ImageQuant software (Amersham).

Ultraviolet sensitivity
Survival assays were performed as described (53Go). Briefly, 5x105 cells were plated per well in six well dishes, grown for 2 days until confluent and irradiated with UV at 254 nm in a FB-UVXL-1000 UV crosslinker (Fisher) under 3 ml of medium to prevent dehydration. Cells were trypsinized, and 5x105 cells were re-plated and allowed to recover for 2 days. Survival was determined by trypsinization, trypan blue staining and counting using a hemacytometer.

IR sensitivity
Cells were irradiated with the indicated doses of {gamma}-rays from a 137Cs source, plated in triplicate (800–8000 cells/10 cm dish) and cultured for 7 days. After methylene blue staining, percentage survival was determined by colony number of treated cells normalized to unexposed cells.

S-phase checkpoint assay
Inhibition of DNA synthesis after UV or {gamma} radiation was assessed as described previously (54Go). Cells were pre-labeled with 25 nCi of 14C thymidine (NEN) per ml for 24 h, irradiated, incubated for 1 h and pulse labeled with 5 uCi/ml of 3H-thymidine (NEN). Cells were harvested and radioactivity quantitated in a liquid scintillation counter. The measure of DNA synthesis inhibition was derived from resulting ratios of 3H to 14C counts.

G2/M checkpoint
For immunofluorescent detection of phospho-histone H3, cells fixed in 70% ethanol were resuspended in 0.25% Triton X-100 in PBS and incubated on ice for 15 min, then room temperature for 3 h in PBS containing 1% BSA and anti-phospho-histone H3 antibody (Cell Signaling Technology) followed by FITC-conjugated anti-mouse secondary antibody. Cellular fluorescence was detected on a FACScan flow cytometer (55Go).

Chromosome analyses
Cells were arrested in metaphase using a 1 h incubation with colcemid (100 ng/ml; GIBCO/BRL; KaryoMAX solution) and metaphases were prepared as described (41Go). For UV treatment prior to chromosome analysis, near confluent cells were irradiated as described earlier in a 10 cm dish containing 10 ml of media and allowed to recover for 24 or 48 h. Chromosomal aberrations were quantified using an Olympus BX-61 microscope equipped with an Applied Spectral Imaging interferometer and 40x and 63x objectives.


   ACKNOWLEDGEMENTS
 
We thank Dr Chongzhi Guo for advice on RNAi and Drs JoAnn Sekiguchi, John Moran, Mats Ljungman and Thomas Glover for helpful discussions and critical reading of the manuscript. This work was supported by grants to D.O.F. from the Sidney Kimmel Foundation, the NIH (R01 HL079118) and the Munn endowment of the University of Michigan Comprehensive Cancer Center. Funding to pay the Open Access publication charges for this article was provided by the Sidney Kimmel Cancer Research Foundation.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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