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Molecular Cancer Research 1:207-218 (2003)
© 2003 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

DNA Replication-Dependent Nuclear Dynamics of the Mre11 Complex1

Olga K. Mirzoeva1 and John H.J. Petrini1,2

1 Laboratory of Genetics, University of Wisconsin Medical School, Madison, WI and
2 Molecular Biology Program, Memorial Sloan Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY

Requests for reprints: John H.J. Petrini, Memorial Sloan Kettering Cancer Center, RRL 901B, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-2927; Fax: (646) 422-2062. E-mail: petrinij{at}mskcc.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The Mre11 complex undergoes dramatic relocalization in the nuclei of {gamma}-irradiated and replicating human cells. In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization. The data indicate that the complex is deposited on chromatin in an S phase-specific manner. Mre11 complex chromatin association in S phase was resistant to detergent extraction, in contrast to that in {gamma}-irradiated cells. The complex exhibits extensive colocalization with proliferating cell nuclear antigen throughout S phase, and chromatin loading is enhanced by replication fork stalling, suggesting that the replication fork is a site of Mre11 complex chromatin loading. This is supported by the observation that the complex localized to single-stranded DNA arising in hydroxyurea-treated cells. Although the Mre11 complex appears to function as a DNA damage sensor, limited colocalization with Brca1 or {gamma}-H2AX was observed, arguing that neither DNA damage nor {gamma}-H2AX is required for Mre11 complex chromatin loading. These data provide a potential molecular basis for promotion of sister chromatid association and recombination by the Mre11 complex as well as for ATM-Mre11 complex-dependent activation of cell cycle checkpoints.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The interdependence of DNA replication and DNA recombination is well established. The Mre11 complex (Mre11, Rad50, and Nbs1 in mammals; Xrs2 is the third member in Saccharomyces cerevisiae) is a highly conserved protein complex that mediates diverse functions in DNA recombination (reviewed in Refs. 1, 2). Cytologic analysis in mammals and yeast has also demonstrated that the complex binds to DNA double-strand breaks (DSBs) soon after they have formed (3–6), implicating it in DNA damage detection. Several observations link Mre11 complex functions to DNA replication. First, inherited mutations in Nbs1 and Mre11 result in the chromosome instability syndromes Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (A-TLD) (7–9). Although clinically distinct, cells established from NBS and A-TLD patients exhibit similar DNA damage checkpoint defects during S phase (9–11). Analogous S phase checkpoint functions for the S. cerevisiae Mre11 complex have also been established (12–14).

Second, depletion of Mre11 from DT-40 chicken cells or Xenopus laevis extracts results in extensive chromosome breakage on DNA replication (15, 16). The metabolism of DNA secondary structures, presumably arising at the replication fork, is at least partially dependent on the Mre11 complex homologues in Escherichia coli and S. cerevisiae (17–19). Finally, cytologic analysis has shown that the Mre11 complex becomes avidly associated with chromatin and exhibits extensive colocalization with proliferating cell nuclear antigen (PCNA) in cells undergoing DNA synthesis (20). In this setting, the nuclear disposition of the Mre11 complex is similar to that seen in cells treated with ionizing radiation (IR) (6).

In both mammalian and yeast systems, Mre11 complex mutations that confer checkpoint defects also diminish or abrogate detectable DNA damage association, circumstantially linking the two functions, and supporting the interpretation that the complex is involved in sensing DNA damage (8, 9, 21). We have shown that the dynamic behavior of the complex either after IR or in S phase does not depend on ATM (6, 22), consistent with the idea that with regard to DNA damage association, the complex is upstream of ATM.

The basis for Mre11 complex association with chromatin during S phase progression is unclear. To define the molecular correlates of this association, we have examined the Mre11 complex in synchronous cultures of human cells using both cytologic and biochemical approaches. The data suggest that to a limited extent, the complex was associated with stalled replication forks. However, despite similar cytological appearances, the chromatin association established during S phase was qualitatively distinct from that established on IR treatment, arguing that binding to DNA damage is unlikely to constitute the primary mode of Mre11 complex chromatin association in S phase. Rather, we propose that the Mre11 complex is loaded onto chromatin in a DNA replication-dependent manner, and thus plays a structural role in the nascent sister chromatid. These data may provide a basis for interpreting genetic data from S. cerevisiae that implicate the Mre11 complex in facilitating sister chromatid recombination and association, a function that appears to be largely independent of the complex's nuclease activity (23–25).


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
The Mre11 complex exhibits pronounced DNA damage- and cell cycle-dependent changes in its nuclear disposition. Previous data support the interpretation that DNA damage-dependent changes at early time points following IR are attributable to association of the complex with DSBs (5, 6), and it has been suggested that DSB association is influenced by {gamma}-H2AX phosphorylation (26, 27). From the perspective of immunofluorescence analysis, changes in Mre11 complex localization during S phase are virtually indistinguishable from that seen in {gamma}-irradiated cells (22). This observation is somewhat paradoxical, because it is extremely unlikely that DSBs formed during S phase progression are as abundant as those formed in response to the doses of IR typically used. To address this issue, we assessed the molecular correlates of S phase-dependent Mre11 complex dynamics.

Three Distinct Patterns of Mre11 Complex Localization
Asynchronous cultures of primary human fibroblasts exhibited three patterns of Mre11 complex localization following detergent extraction of nucleoplasmic protein: I—PML body-associated pattern; II—microgranulated pattern; and III—negative for the Mre11 staining (Fig. 1A). We previously established that unirradiated cells exhibiting pattern II were in S phase by detection of BrdUrd incorporation and retention of PCNA (6, 22). To confirm this interpretation, Mre11 complex localization was assessed in synchronized cells. PML body staining (pattern I) was seen in 35–50% of 37Lu cells enriched in G1, whereas 35% were negative for Mre11 complex staining, as were mitotic cells in asynchronous cultures. The microgranulated pattern (pattern II) was observed in 50–65% of S phase cells (Fig. 1B). As previously shown in asynchronous cells (22), the Mre11 complex colocalized extensively with PCNA in synchronous S phase cultures (data not shown). Two to five percent of cells with pattern II were PCNA-negative (data not shown), perhaps indicating a fraction of cells with extensive spontaneous DNA damage.



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  		FIGURE 1. Mre11 localization is cell cycle dependent. A. Typical patterns of localization of chromatin-associated fraction of Mre11 complex in asynchronous 37Lu cells: pattern I—PML-body associated, pattern II—microgranulated, and pattern III—negative. m, mitotic cell. B. Distribution of Mre11 localization patterns in synchronized 37Lu cells in G1, G1-S (arrested by mimosine), early, mid-, and late S (3, 6, and 9 h release after mimosine arrest, respectively). Bar, percentage of at least 200 nuclei.

 
Mre11 Complex Chromatin Association: Distinct Outcomes in DNA Replication and {gamma}-Irradiated Cells
To detect Mre11 patterns in S phase, cells were extracted to remove nucleoplasmic proteins and retain those bound to chromatin with greater avidity (6, 22). Pattern II Mre11 staining in S phase is indistinguishable from pattern II staining in {gamma}-irradiated cells. We asked whether the behavior of Mre11 complex in more extensive fractionation of chromatin would distinguish {gamma}-irradiated cells with type II staining from cells in S phase. HeLa cells were synchronized at the G1-S boundary with mimosine and fractionated into the soluble (100 mM NaCl and 0.5% Triton X-100) and insoluble or chromatin-associated (50 units DNase and 500 mM NaCl) fractions at time points following release into cycle. At each time point, synchrony was assessed by fluorescence-activated cell sorting (FACS) analysis (Fig. 2A).



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  		FIGURE 2. Chromatin association in S phase versus {gamma}-irradiated cells. A. Assessment of cell synchrony in HeLa cells. B. Protein levels of Nbs1, actin, PCNA, and {alpha}-tubulin in soluble and chromatin fractions in HeLa cells in G1, G1-S, early, mid-, or late S (S-3, S-6, and S-9 are 3, 6, and 9 h after release from G1-S arrest, respectively), G2 (13 h after mimosine release), and mitosis; M, nocodazol arrest; asyn, asynchronous. C. Nbs1 retention relative to asynchronous cells. Immunoblots were visualized and quantitated on a STORM instrument. The intensity of Nbs1 staining was normalized to the intensity of actin; a value of 1 was assigned to asynchronous cells. D. Nbs1, actin, PCNA, and {alpha}-tubulin levels in soluble and chromatin fractions of HeLa cells unirradiated (un) or irradiated with 12 Gy of IR and harvested at 45 min, and 2 and 8 h post-IR. Percentage of HeLa cells showing type II Mre11 foci at each time point post-IR is indicated below the corresponding lane on the Western blot.

 
Extracts prepared from synchronized cells were resolved by SDS-PAGE and immunoblotted sequentially with Nbs1, actin, PCNA, and {alpha}-tubulin. The level of soluble Nbs1 was significantly reduced in mid- and late S phase (6 and 9 h after release), while a corresponding increase in the chromatin-associated fraction was observed at these time points. As expected, PCNA exhibited a similar increase in chromatin association in S phase (Fig. 2B). To confirm that the conditions used for extraction distinguished soluble from chromatin-associated proteins, the level of {alpha}-tubulin was monitored, and found only in the soluble fraction throughout the cell cycle. Retention of Nbs1 in the S phase-insoluble (chromatin-associated) fraction was reproducibly 4- to 5-fold increased relative to that in asynchronous cultures (Fig. 2C). Levels were highest in mid- to late S phase and lowest in mitosis and G1. These data indicate that the DNA replication-dependent association of Mre11 complex proteins observed by immunofluorescence reflects an avid interaction with chromatin.

Mre11 chromatin association was qualitatively distinct in {gamma}-irradiated cells. Essentially equivalent levels of Mre11 were detected in the soluble and chromatin-associated fractions of untreated versus {gamma}-irradiated cells from 45 min to 8 h post-IR (Fig. 2D). The formation of type II Nbs1 foci in HeLa cells was monitored and found to be similar to that observed in 37Lu primary fibroblasts (Fig. 2D; percentage of positive nuclei is noted below the corresponding lanes on the Western blot) (6). These data indicate that DNA replication-dependent association of the Mre11 complex is qualitatively distinct from that which occurs in response to DSB formation.

Chromatin Association Is Enhanced by Replication Fork Stalling
The observed colocalization of Mre11 and PCNA throughout S phase suggested that Mre11 complex deposition on chromatin during DNA replication may occur at least partially at replication forks. Because previous studies have established that Mre11 complex foci form in response to replication fork stalling (3, 28), we asked whether Mre11 complex chromatin association would be enhanced in that circumstance. HeLa cells were synchronized with mimosine and treated with 1 mM HU at 3 and 6 h after release from G1-S arrest for 1 h. At 3 h, the level of chromatin-associated Mre11 complex was markedly increased (Fig. 3A), whereas no increase was detectable at 6 h or in asynchronous cells.



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  		FIGURE 3. Mre11 complex chromatin association and replication fork stalling. A. Immunoblots for Nbs1, Ku70, actin, and PCNA in soluble and chromatin fraction of cells after replication arrest. (+) Addition of 1 mM of HU; (-) no treatment. Time points after synchrony are indicated. B. Cell-cycle status of cells in the presence or absence of HU determined by FACS. C. Colocalization of Mre11 and ssDNA induced by HU treatment. Cell costained with Mre11 antiserum (red) and BrdUrd monoclonal antibody (mAb) (green) is shown. I, enlarged inset of the merged image; II, the same inset with green channel offset by five pixels to the right.

 
The increased chromatin association was correlated with Mre11 complex localization to stalled replication forks. 37Lu cells were grown in the presence of BrdUrd, synchronized in early or mid-S phase, and treated with 1 mM HU for 1 h (Fig. 3B). BrdUrd-substituted ssDNA forming at stalled replication forks was detected by immunofluorescence with a BrdUrd mAb as described (29). BrdUrd staining was absent in untreated cells. In contrast, 20% of HU-treated cells exhibited BrdUrd foci, which colocalized extensively with Mre11 foci (Fig. 3C). Colocalization was readily evident by comparing inset I to inset II in which the green channel (BrdUrd staining) was offset by five pixels to the right (Fig. 3C, compare insets I and II).

S Phase Chromatin Association Is Not DNA Damage Dependent
Given its previously established role in DSB detection, we examined the possibility that loading of the Mre11 complex onto chromatin may occur at DSBs arising in S phase. The histone variant, H2AX, is rapidly phosphorylated on the induction of DNA damage; the phosphorylated H2AX is designated {gamma}-H2AX (30, 31). We reasoned that if Mre11 complex chromatin association were dependent on DSBs, this would be reflected by colocalization of Mre11 and {gamma}-H2AX foci in S phase cells.

First, the appearance of {gamma}-H2AX in undamaged, detergent-extracted cells was assessed. Approximately 75% of asynchronous cells were negative for {gamma}-H2AX, whereas 15–20% exhibited {gamma}-H2AX foci which were variable in size and intensity (patterns I and III; Fig. 4A), in agreement with previous reports (4). These {gamma}-H2AX-positive cells also retained PCNA, which, under the extraction conditions used, indicated that they were in S phase (22, 32). A minor fraction of the asynchronous population contained several large foci per nucleus (pattern II), and were predominantly PCNA-negative (Fig. 4A).



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  		FIGURE 4. {gamma}-H2AX dynamics in replicating cells. A. Double immunostaining with {gamma}-H2AX antiserum (red) and PCNA mAb (green) in asynchronous 37Lu cells. {gamma}-H2AX patterns are designated as I—bright whole nucleus, II—foci of different sizes and intensities, and III—several large foci as in "Results." B. Distribution of {gamma}-H2AX patterns in cells synchronized at G1, G1-S (mimosine arrest), early, mid-, and late S phase (3, 6, and 9 h after release from mimosine arrest, respectively). Bar, percentage of at least 200 nuclei counted.

 
To confirm that H2AX phosphorylation was induced in S phase cells, {gamma}-H2AX patterns were assessed at time points after release from mimosine-induced G1-S arrest. The percentage of nuclei with patterns I and II did not vary significantly with entry and progression through S phase. However, pattern III was observed in approximately 50% of S phase nuclei, representing a 3-fold increase over G1-S or asynchronous cells (Fig. 4B). These data indicated that H2AX phosphorylation was induced during the course of DNA replication.

The observation of {gamma}-H2AX formation in DNA replication is consistent with recent findings in cells treated with HU or UV light (3, 28, 33), which correlate H2AX phosphorylation with the inhibition of replication fork progression. We found that {gamma}-H2AX foci in untreated S phase cells colocalized with PCNA in early S phase. In mid-S phase, when both PCNA and {gamma}-H2AX foci were present at maximal levels, colocalization was not observed. Both PCNA and {gamma}-H2AX foci were markedly reduced in late S phase, but limited colocalization was evident (Fig. 5). Although PCNA loads at replication forks, it accumulates on chromatin after the fork has progressed (34). Therefore, the relative decrease in PCNA-{gamma}-H2AX colocalization in later S phase time points may indicate that {gamma}-H2AX does not colocalize with PCNA away from the replication fork. The data obtained are consistent with the interpretation that {gamma}-H2AX forms at the replication fork, conceivably as a result of DSB formation as well as in response to the presence of DNA replication intermediates.



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  		FIGURE 5. {gamma}-H2AX and PCNA. Representative pictures of 37Lu cells synchronized at early, mid-, and late S phase immunostained with {gamma}-H2AX antiserum (red) and PCNA mAb (green). Enlarged insets from the merged images are shown at right.

 
We assessed the degree to which Mre11 foci colocalized with {gamma}-H2AX in synchronous 37Lu cultures. As with {gamma}-H2AX and PCNA, {gamma}-H2AX and Mre11 exhibited partial colocalization early in S phase, and the degree of colocalization in mid-S phase was markedly reduced. As with PCNA, Mre11 became localized to fewer, but larger foci in late S phase, and limited colocalization with {gamma}-H2AX was observed (Fig. 6). In addition {gamma}-H2AX and Mre11 foci did not colocalize in the rare non-S phase cells exhibiting {gamma}-H2AX pattern II (Fig. 6, bottom row).



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  		FIGURE 6. Mre11 and {gamma}-H2AX foci in S phase cells. Representative pictures of 37Lu synchronized at early, mid-, and late S phase and immunostained with {gamma}-H2AX antiserum (red), Mre11 mAb 8F3 (green) and merged are shown. Rare non-S phase cells with Mre11 and {gamma}-H2AX foci are presented in the bottom row. Enlarged insets from the merged images are shown at right.

 
{gamma}-H2AX and Mre11 colocalization in IR-treated cells has not been examined under the conditions employed in this study. Previous data from nonextracted cells demonstrated colocalization of {gamma}-H2AX and the Mre11 complex in IR-induced foci at 4–8 h post-IR (4). We asked whether {gamma}-H2AX and Mre11 foci colocalized at early times after IR treatment using in situ cell fractionation (6). Mre11 and {gamma}-H2AX exhibited very different patterns of localization 1 and 2 h post-IR (Fig. 7 and data not shown) and become significantly coincident at late time (8 h post-IR). Thus, at early time points after IR, {gamma}-H2AX and Mre11 foci were independent, consistent with previous data (4).



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  		FIGURE 7. Mre11 and {gamma}-H2AX foci in {gamma}-irradiated cells. 37Lu cells were irradiated with 12 Gy and stained with 8F3 (green) or {gamma}-H2AX (red) 1 or 8 h post-IR.

 
Collectively, these data suggest that {gamma}-H2AX and Mre11 localize to distinct structures in replicating or damaged cells. To a limited extent, Mre11 and {gamma}-H2AX may localize at DSB sites or DNA replication forks in S phase. However, the great majority of replication-specific Mre11 foci do not appear to represent either DSB sites or sites of DNA repair.

S Phase Mre11 and Brca1 Foci
Changes in the disposition of Brca1 are also associated with DNA replication. Brca1 forms foci in S phase cells and these foci relocalize in response treatment with HU or IR to become partially colocalized with PCNA (28, 35). On this basis, it has been suggested that Brca1 may influence recombinational repair of replication forks, consistent with other evidence implicating it in DNA recombination (36, 37). Because Brca1 physically interacts with the Mre11 complex, and forms foci that colocalize in {gamma}-irradiated cells (28, 38), we asked whether Brca1 and Mre11 complex foci colocalized in S phase.

37Lu cells were synchronized by mimosine and released at different stages of S phase after mimosine block. Some degree of colocalization was evident throughout S phase, particularly in early S, but Mre11 and Brca1 generally exhibited distinct patterns of localization at later time points in S phase (Fig. 8). A similar outcome was observed in asynchronous cultures where the extent of S phase progression was deduced from Mre11 (equivalent to PCNA) patterns. Thus, the Mre11 complex's S phase chromatin association is not coincident with that of Brca1, although in HU or UV-treated cells, both appear to bind stalled replication forks (Fig. 3C) (3, 28, 35). This further supports the interpretation that the Mre11 complex chromatin association is not dependent on DNA damage.



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  		FIGURE 8. S-phase Mre11 and Brca1 foci. Representative images of 37Lu cells at various stages of S phase immunostained with Mre11 antiserum (red) and Brca1 mAb (green), and merged.

 
Mre11 Complex Chromatin Association Does Not Require ATM or ATR
In yeast and humans, members of the Mre11 complex are phosphorylated by ATM and ATR or their orthologues in response to DNA damage (12–14, 39–42). We addressed whether modifications of the Mre11 complex or other ATM/ATR targets were required for S phase or IR-induced chromatin association.

We previously established that neither IR-induced nor S phase Mre11 foci required ATM activity (6, 22). To inhibit both ATR and ATM, 37Lu cells were treated with 10 mM caffeine for varying times before {gamma}-irradiation. This concentration of caffeine effectively inhibits ATM and ATR, and reduces DNA-PK activity by approximately 50% in vitro (43). Cells were pretreated with caffeine for 70, 40, or 11 min before {gamma}-irradiation and Mre11 foci were assessed in in situ fractionated cells 2 h later. Neither S phase nor IR-induced foci were affected (Table 1. To control for the effectiveness of caffeine treatment, cells were also scored for entry into mitosis, a transition that is blocked by the caffeine-sensitive G2-M checkpoint. Entry into mitosis was indicated by the presence of phosphorylated histone H3. At each time point, abrogation of the G2-M checkpoint was clearly evident, indicating that caffeine treatment was effective (data not shown). These data argue that Mre11 complex chromatin association does not require ATM or ATR activity, and are consistent with the interpretation that the association in S phase does not reflect the metabolism of DNA damage.


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  		Table 1. Effect of Caffeine Treatment of Mre11 Complex Focus Formation

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this study, we show that the Mre11 complex is deposited on chromatin in an S phase-specific manner. The chromatin association established during DNA replication is qualitatively distinct from that in {gamma}-irradiated cells; the Mre11 complex is significantly more abundant on chromatin in S phase cells and is resistant to extraction with nonionic detergent. The association is enhanced by HU treatment, suggesting that loading of Mre11 complex proteins occurs, at least in part, at the replication fork. Consistent with this possibility, the complex localizes to regions of ssDNA appearing in response to replication fork arrest. In addition, the Mre11 complex extensively colocalizes with PCNA throughout S phase in asynchronous (22) and synchronous cultures (this study). Despite the fact that the Mre11 complex localizes to DSBs formed in response to {gamma}-irradiation, the data argue that its association with chromatin during S phase does not occur at sites of spontaneous DNA damage. Therefore, these data suggest that some Mre11 complex functions during DNA replication are temporally and spatially distinct from its role in DSB metabolism.

The functional significance of the Mre11 complex's association with nascent chromatids is not clear. The Mre11 complex has been shown to influence transitions in chromatin structure before the initiation of meiotic recombination (44). As shown here for the human complex, the S. cerevisiae Mre11 complex associates with chromatin during late premeiotic S phase; loading is dependent on the activity of the B-type cyclins Clb5 and Clb6 which are absolutely required for premeiotic replication.2 Thus, it is conceivable that establishment of Mre11 association during DNA replication reflects a general function in modulating chromatin structure.

On the basis of the cell cycle dependence of Mre11 complex mutant phenotypes, it has been suggested that the Mre11 complex influences sister chromatid association. Mre11 complex-deficient S. cerevisiae strains are highly sensitive to clastogens (20). In particular, IR sensitivity of synchronized haploid and diploid mre11{Delta} strains is approximately 70-fold higher in G2 than a synchronous wild-type strain (25). However, S. cerevisiae Mre11 complex mutants exhibit increased spontaneous interhomologue recombination frequencies (20), indicating that Mre11 deficiency differentially impairs sister chromatid recombination. Similarly, Schizosaccharomyces pombe rad50{Delta} mutants exhibit 28-fold increased interhomologue recombination rates, whereas sister chromatid recombination is 15-fold reduced (45). Nonhomologous end-joining defects in S. cerevisiae rad50{Delta} strains are most pronounced in non-G1 cells, indicating that Mre11 complex functions are linked to the presence of sister chromatids even when they are not templates for homologous recombination (24). Collectively, these data suggest the Mre11 complex primarily influences sister chromatid association and recombination. Deposition of the complex on newly synthesized chromatids would be a logical prerequisite to such a function.

Data from vertebrate systems are consistent with this proposal. Sister chromatid breaks accumulate in Mre11-depleted DT-40 cells and X. laevis in vitro replication reactions, indicating that DSBs formed during DNA replication are poorly metabolized (15, 16). Interestingly, the S. pombe rad50{Delta} is epistatic to a hypomorphic rad21 (SCC1) mutation for MMS sensitivity (45), supporting the hypothesis that the Mre11 complex and sister chromatid cohesion functions are linked. In this context, we propose that the cytologic behavior of the human Mre11 complex reflects its role in promoting sister chromatid association and recombination.

Structural analyses of the Mre11 complex provide a molecular basis for its role in facilitating chromatid interactions. Scanning force microscopy reveals that complexes of human Mre11 and Rad50 bind DNA via a globular domain, while the coiled coil regions of Rad50 form an extended intramolecular flexible arm. Thus, interactions between the coiled coils of DNA-bound Mre11 complexes would tether sister chromatids or DNA ends within the same chromatid (46). Crystallographic analysis of Pyrococcus furiousis Mre11 complex has identified a conserved "hook" motif within the coiled coil domain of Rad50 that could mediate such an interaction. This motif contains two cysteine residues, which when complexed with a second hook region, coordinate a Zinc atom and form an interlocking hook with the intramolecular coiled coil Rad50 arms extending outward in opposite polarities (47). These structural data provide a molecular basis for the Mre11 complex to facilitate sister chromatid interaction.

In addition to reflecting a role in DSB repair, the complex's chromatin association may be linked to the activation of the intra-S phase checkpoint. Mre11 complex nuclear localization is dramatically reduced in NBS and A-TLD cells, both of which exhibit intra-S phase checkpoint defects (8, 9, 21). Accordingly, S phase Mre11 complex foci are nearly absent in those settings (22) (data not shown). The sister chromatid cohesion protein, SMC-1, which, like the Mre11 complex, is loaded onto chromatin during S phase, is phosphorylated by ATM in response to DNA damage (48–50). This phosphorylation event is required for intra-S phase checkpoint activation, and is abrogated in NBS cells (49, 50). Hence, Mre11 complex chromatin association may be a prerequisite for linking ATM activation to SMC-1. An additional possibility is that Mre11 complex chromatin association facilitates its own phosphorylation by ATM, an event also required for intra-S phase checkpoint activation (51, 52). Consistent with this possibility, neither IR-induced nor S phase Mre11 complex foci requires ATM (6, 22), indicating that chromatin-bound Nbs1 is the likely substrate of ATM.

What is responsible for recruiting the Mre11 complex to chromatin? It appears that loading in S phase can occur at the replication fork; whether this constitutes the primary mode of chromatin association remains an open question. It has been proposed that {gamma}-H2AX recruits the Mre11 complex to damaged DNA (4, 53). Data presented here appear to contradict that interpretation. Mre11 complex loading in S phase or after {gamma}-irradiation was unaffected by caffeine treatment, and is unaffected by ATM deficiency (6, 22) or ATR depletion;3 these are experimental contexts in which H2AX phosphorylation would be reduced (33, 54).4 Moreover, we failed to detect significant colocalization of Mre11 and {gamma}-H2AX in S phase or {gamma}-irradiated cells. Celeste et al. (26) observed S phase Mre11 complex foci in in situ fractionated H2AX-deficient cells, supporting the conclusion that {gamma}-H2AX does not recruit the Mre11 complex during DNA replication. In that study, Mre11 complex focus formation was found to be suppressed in {gamma}-irradiated H2AX-deficient cells. However, this assessment was made 8 h post-IR without in situ fractionation, conditions in which Mre11 foci are substantially reduced in wild-type human cells (6, 53, 55). Although we cannot exclude the possibility that {gamma}-H2AX recruits the Mre11 complex via an interaction too short-lived to be detected, the data presented here from in situ fractionated cells at early time points are most consistent with the interpretation that, as in S phase cells, {gamma}-H2AX is not required for Mre11 complex chromatin association in {gamma}-irradiated cells. This issue would be most convincingly addressed in a phosphorylation site mutant of H2AX. Whatever its molecular determinants may be, chromosome instability and impaired checkpoint functions are correlated with mutations that reduce Mre11 complex chromatin association. This underscores the importance of this previously undescribed aspect of Mre11 complex dynamics.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Lines
HeLa cells and 37Lu normal diploid fibroblasts were obtained from the American Type Culture Collection. 37Lu cells were used at passages 7–12. The cells were grown at 37°C in 5% CO2 in DMEM with 10% FCS and were negative in monthly Mycoplasma tests.

Cell Cycle Analysis
Cells were washed twice with PBS and refed with DMEM containing 0.1% FCS. After 2 days, the media were changed to DMEM with 10% FCS and 200 µM mimosine for 24 h to arrest the cells at G1-S. Arrested cells were washed twice and refed with DMEM containing 10% FCS for 3 h (early S), 6 h (mid-S), 9 h (late S), and 13 h (G2). Cells in mitosis (M) were synchronized by culturing in the presence of 50 ng/ml nocodazol for 16 h and mitotic cells were shaken off the dishes. Cells in G1 were obtained 5 h after release from nocodazol arrest by washing and culture in normal media.

For FACS analysis, 106 cells were fixed in ice-cold 70% ethanol for at least 1 h at -20°C, and stained with propidium iodide [30 µg/ml and RNase 0.6 mg/ml in PBS + 0.5% (v/v) Tween 20 + 0.1% (w/v) BSA]. Stained cells were analyzed on a Becton-Dickinson FACScan.

Chromatin Fractionation
Typically, 107 cells were used for each extraction. Cells were harvested by trypsinization, resuspended in growth media, and counted. Cells were lysed in ice-cold CSK buffer [10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM ATP, 10 mM sodium ß-glycerophosphate, 10 mM sodium-orthovanadate] for 10 min at 2.5 x 107 cells/ml. The supernatant from low-speed centrifugation (5 min, 1500 x g, 4°C) constituted the soluble fraction that was subsequently clarified by high-speed centrifugation (10 min at 16,000 x g, 4°C). The low-speed pellet, consisting of intact nuclei (verified microscopically), was washed once with ice-cold CSK buffer (5 min, 1500 x g, 4°C) and incubated in CSK buffer containing 50 units of RNase-free DNase I (Roche Biochemicals, Basel, Switzerland) at 1 x 108 nuclei/ml for 30 min at 37°C. DNase-treated nuclei were collected by centrifugation (10 min, 16,000 x g, 4°C) and the supernatant was retained. The DNase pellet was washed once in 1 ml of ice-cold CSK buffer (5 min, 16,000 x g, 4°C) and incubated in ice-cold CSK buffer containing 500 mM NaCl for 10 min at 4°C. This extract was clarified by centrifugation (10 min, 16,000 x g, 4°C) and pooled with the DNase-treated fraction, constituting the chromatin-associated, or insoluble, fraction. Protein extracts were quantitated by Bio-Rad DC protein concentration assay and 25 µg for soluble fraction and 5 µg for insoluble were resolved by 8% SDS-PAGE and Western blotted as described previously (8). Equal loading and transfer was monitored by Ponceau red staining of the membrane and actin immunoblotting. The membranes were immunoblotted subsequently with antibodies to Nbs1, Mre11, Ku70, {alpha}-tubulin (as a fractionation control), and PCNA (as cell cycle and fractionation control), and visualized by ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ). For quantification, blots were phosphorimaged with a STORM scanner and analyzed using ImageQuant software (Molecular Dynamics, Piscataway, NJ); abundance is expressed relative to values obtained from asynchronous cells.

Immunofluorescence
37Lu cells were grown on 18 mm coverslips for 48 h or synchronized as above. Before fixation, in situ fractionation was performed as described (6). The cells were fixed in modified Streck tissue fixative for 30 min at room temperature and permeabilized for 15 min at room temperature as described (5). Cells were blocked with 10% FCS in PBS then stained with primary antibody in PBS with 5% FCS for 1 h at room temperature; secondary antibody staining was 30 min. DAPI counterstain was used at a final concentration of 0.05 µg/ml in the last wash. Controls with preimmune serum or secondary antibody alone gave no signal. Images were captured with a CCD camera (Princeton Instruments, Trenton, NJ). Gray scale images were processed by using IP Labs software (Signal Analytics Corporation, Vienna, VA) and Photoshop 6 (Adobe, San Jose, CA).

HU Treatment and Detection of Single-Stranded DNA Breaks
One millimolar HU was added to asynchronous or synchronized HeLa cells for 1 h. Treated cells were harvested, fractionated, and used for Western blot as described above. For immunofluorescent detection of ssDNA, 37Lu cells were grown on coverslips to 70% confluence, incubated in DMEM + 0.1% FCS for 24 h, 30 µM BrdUrd was added, and the cells were cultured for 24 h. Media were replaced with DMEM + 10% FCS, 200 µM mimosine, and 30 µM BrdUrd for 24 h. Cells were released from G1-S arrest into mimosine-free media for the indicated times then BrdUrd was washed off and 1 mM HU was added to the growth media for 1 h. ssDNA was visualized as described (29) using a BrdUrd mAb which is specific for BrdUrd-substituted ssDNA.

Caffeine Treatment and Detection of G2-M Checkpoint
37Lu cells were plated in triplicate onto 18 mm coverslips at 5000 cells/coverslip and cultured for 2 days. DMEM + 10% FCS with 10 mM caffeine was added to the cells 70, 40, or 11 min before {gamma}-irradiation. The cells were subsequently {gamma}-irradiated at 12 Gy in a Mark I137Cs source at 2.5 Gy/min or mock treated, allowed to recover for 2 h, in situ fractionated as described (6), and stained with Mre11 antiserum. For detection of mitosis, the cells were fixed in 4% paraformaldehyde without prior extraction and permeabilized in Triton X-100 solution [100 mM Tris-HCl (pH 7.4), 50 mM EDTA, 0.5% Triton X-100]. The cells were immunostained with phospho-histone H3 (mitotic marker) antibody, followed by FITC-conjugated secondary antibody and counterstained with DAPI. The total amount of mitotic cells at each coverslip was counted for unirradiated and irradiated cells in the presence or absence of caffeine.

Immunological Reagents
Nbs1 (#16), Mre11 (#59) antisera, and Mre11 mAb 8F3 were described previously (8,9). {gamma}-H2AX and anti-Histone H3 antisera were from Upstate Biotechnology, Waltham, MA, Brca1 mAb (MS110) was from Calbiochem, La Jolla, CA, {alpha}-tubulin mAb (B512) and actin mAb (clone AC 40) were from Sigma, St. Louis, MO, PCNA mAb (PC10) was from Santa Cruz, Santa Cruz, CA, BrdUrd mAb (3D4) was from PharMingen, San Diego, CA, and Ku70 mAbs (N3H10) were obtained from R. Burgess, UW-Madison, WI. All secondary antibodies were from Jackson Immunoresearch Laboratories.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
The authors are grateful to the members of the Petrini Lab for insights throughout the course of this study and on the manuscript, to Andy Koff for comments and suggestions on the manuscript, to W. Bonner for {gamma}-H2AX antiserum, and to Jan Karlseder and Titia de Lange for advice and reagents.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH GM56888 and the DOE ER62859. Back

2 Borde et al., manuscript in preparation. Back

3 Brown and Baltimore, unpublished. Back

4 Karlseder et al. Repression of the DNA damage response by the telomeric protein TRF2, submitted for publication. Back

Received September 9, 2002; revised November 12, 2002; accepted November 27, 2002.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Petrini, J. H. J. S phase functions of the Mre11 complex. Cold Spring Harbor Symp. Quant. Biol., 65: 405–412, 2000.[Medline]
  2. Haber, J. E. The many interfaces of Mre11. Cell, 95: 583–586, 1998.[Medline]
  3. Limoli, C. L., Giedzinski, E., Bonner, W. M., and Cleaver, J. E. UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, {gamma}-H2AX formation, and Mre11 relocalization. Proc. Natl. Acad. Sci. USA, 99: 233–238, 2002.[Abstract/Free Full Text]
  4. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol., 10: 886–895, 2000.[Medline]
  5. Nelms, B. E., Maser, R. S., MacKay, J. F., Lagally, M. G., and Petrini, J. H. J. In situ visualization of DNA double-strand break repair in human fibroblasts. Science, 280: 590–592, 1998.[Abstract/Free Full Text]
  6. Mirzoeva, O. K. and Petrini, J. H. DNA damage-dependent nuclear dynamics of the mre11 complex. Mol. Cell. Biol., 21: 281–288, 2001.[Abstract/Free Full Text]
  7. Varon, R., Vissinga, C., Platzer, M., Cerosaletti, K. M., Chrzanowska, K. H., Saar, K., Beckmann, G., Seemanova, E., Cooper, P. R., Nowak, N. J., Stumm, M., Weemaes, C. M., Gatti, R. A., Wilson, R. K., Digweed, M., Rosenthal, A., Sperling, K., Concannon, P., and Reis, A. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell, 93: 467–476, 1998.[Medline]
  8. Carney, J. P., Maser, R. S., Olivares, H., Davis, E. M., Le Beau, M., Yates, J. R., III, Hays, L., Morgan, W. F., and Petrini, J. H. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell, 93: 477–486, 1998.[Medline]
  9. Stewart, G. S., Maser, R. S., Stankovic, T., Bressan, D. A., Kaplan, M. I., Jaspers, N. G., Raams, A., Byrd, P. J., Petrini, J. H., and Taylor, A. M. The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell, 99: 577–587, 1999.[Medline]
  10. Taalman, R. D., Jaspers, N. G., Scheres, J. M., de Wit, J., and Hustinx, T. W. Hypersensitivity to ionizing radiation, in vitro, in a new chromosomal breakage disorder, the Nijmegen breakage syndrome. Mutat. Res., 112: 23–32, 1983.[Medline]
  11. Lavin, M. F. and Shiloh, Y. The genetic defect in ataxia-telangiectasia. Annu. Rev. Immunol., 15: 177–202, 1997.[Medline]
  12. Usui, T., Ogawa, H., and Petrini, J. H. A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell, 7: 1255–1266, 2001.[Medline]
  13. Grenon, M., Gilbert, C., and Lowndes, N. F. Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol., 3: 844–847, 2001.[Medline]
  14. D'Amours, D. and Jackson, S. P. The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev., 15: 2238–2249, 2001.[Abstract/Free Full Text]
  15. Costanzo, V., Robertson, K., Bibikova, M., Kim, E., Grieco, D., Gottesman, M., Carroll, D., and Gautier, J. Responses to DNA damage in Xenopus: Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. Mol. Cell, 8: 137–147, 2001.[Medline]
  16. Yamaguchi-Iwai, Y., Sonoda, E., Sasaki, M. S., Morrison, C., Haraguchi, T., Hiraoka, Y., Yamashita, Y. M., Yagi, T., Takata, M., Price, C., Kakazu, N., and Takeda, S. Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells. EMBO J., 18: 6619–6629, 1999.[Medline]
  17. Connelly, J. C., Kirkham, L. A., and Leach, D. R. The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proc. Natl. Acad. Sci. USA, 95: 7969–7974, 1998.[Abstract/Free Full Text]
  18. Leach, D. R. Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair. BioEssays, 16: 893–900 issn: 0265–9247, 1994.[Medline]
  19. Lobachev, K. S., Gordenin, D. A., and Resnick, M. A. The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell, 108: 183–193, 2002.[Medline]
  20. Maser, R. S., Bressan, D. A., and Petrini, J. H. J. The Mre11-Rad50 complex: diverse functions in the cellular DNA damage response. In: M. F. Hoekstra and J. A. Nickoloff (eds.), DNA Damage and Repair, pp. 147–172, vol. III. Totowa: Humana Press, 2001.
  21. Williams, B. R., Mirzoeva, O. K., Morgan, W. F., Lin, J., Dunnick, W., and Petrini, J. H. A murine model of Nijmegen breakage syndrome. Curr. Biol., 12: 648–653, 2002.[Medline]
  22. Maser, R. S., Mirzoeva, O. K., Wells, J., Olivares, H., Williams, B. R., Zinkel, R., Farnham, P. J., and Petrini, J. H. J. The MRE11 complex and DNA replication: linkage to E2F and sites of DNA synthesis. Mol. Cell. Biol., 21: 6006–6016, 2001.[Abstract/Free Full Text]
  23. Ivanov, E. L., Korolev, V. G., and Fabre, F. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. Genetics, 132: 651–664, 1992.[Abstract]
  24. Moore, J. K. and Haber, J. E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol., 16: 2164–2173, 1996.[Abstract]
  25. Bressan, D. A., Baxter, B. K., and Petrini, J. H. The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol., 19: 7681–7687, 1999.[Abstract/Free Full Text]
  26. Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M. J., Redon, C., Pilch, D., Olaru, A., Eckhaus, M., Camerini-Otero, D., Tessarollo, L., Livak, F., Manova, K., Bonner, W. M., Nussenzweig, M. C., and Nussenzweig, A. Genomic instability in mice lacking histone H2AX. Science, 296: 922–927, 2002.[Abstract/Free Full Text]
  27. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol., 10: 886–895, 2000.
  28. Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S. J., and Qin, J. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev., 14: 927–939, 2000.[Abstract/Free Full Text]
  29. Raderschall, E., Golub, E. I., and Haaf, T. Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage. Proc. Natl. Acad. Sci. USA, 96: 1921–1926, 1999.[Abstract/Free Full Text]
  30. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem., 273: 5858–5868, 1998.[Abstract/Free Full Text]
  31. Rogakou, E. P., Boon, C., Redon, C., and Bonner, W. M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol., 146: 905–916, 1999.[Abstract/Free Full Text]
  32. Kill, I. R., Bridger, J. M., Campbell, K. H., Maldonado-Codina, G., and Hutchison, C. J. The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J. Cell Sci., 100: 869–876, 1991.[Abstract]
  33. Ward, I. M. and Chen, J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem., 276: 47759–47762, 2001.[Abstract/Free Full Text]
  34. Tsurimoto, T. PCNA, a multifunctional ring on DNA. Biochim. Biophys. Acta, 1443: 23–39, 1998.[Medline]
  35. Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell, 90: 425–435, 1997.[Medline]
  36. Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. Brca1 controls homology-directed DNA repair. Mol. Cell, 4: 511–518, 1999.[Medline]
  37. Bhattacharyya, A., Ear, U. S., Koller, B. H., Weichselbaum, R. R., and Bishop, D. K. The breast cancer-susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA crosslinking agent cisplatin. J. Biol. Chem., 275: 23899–23903, 2000.[Abstract/Free Full Text]
  38. Zhong, Q., Chen, C. F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z. D., and Lee, W. H. Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. Science, 285: 747–750, 1999.[Abstract/Free Full Text]
  39. Wu, X., Ranganathan, V., Weisman, D. S., Heine, W. F., Ciccone, D. N., O'Neill, T. B., Crick, K. E., Pierce, K. A., Lane, W. S., Rathbun, G., Livingston, D. M., and Weaver, D. T. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature, 405: 477–482, 2000.[Medline]
  40. Zhao, S., Weng, Y. C., Yuan, S. S., Lin, Y. T., Hsu, H. C., Lin, S. C., Gerbino, E., Song, M. H., Zdzienicka, M. Z., Gatti, R. A., Shay, J. W., Ziv, Y., Shiloh, Y., and Lee, E. Y. Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature, 405: 473–477, 2000.[Medline]
  41. Lim, D. S., Kim, S. T., Xu, B., Maser, R. S., Lin, J., Petrini, J. H. J., and Kastan, M. B. ATM phosphorylates p95/Nbs1 in an S-phase checkpoint pathway. Nature, 404: 613–617, 2000.[Medline]
  42. Gatei, M., Young, D., Cerosaletti, K. M., Desai-Mehta, A., Spring, K., Kozlov, S., Lavin, M. F., Gatti, R. A., Concannon, P., and Khanna, K. ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat. Genet., 25: 115–119, 2000.[Medline]
  43. Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M., and Abraham, R. T. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res., 59: 4375–4382, 1999.[Abstract/Free Full Text]
  44. Ohta, K., Shibata, T., and Nicolas, A. Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J., 13: 5754–5763, 1994.[Medline]
  45. Hartsuiker, E., Vaessen, E., Carr, A. M., and Kohli, J. Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair. EMBO J., 20: 6660–6671, 2001.[Medline]
  46. de Jager, M. and Noort, V. Human Rad50/Mre11 is a flexible complex that can tether DNA ends. Mol. Cell, 8: 1129–1135, 2001.[Medline]
  47. Hopfner, K. P., Craig, L., Moncalian, G., Zinkel, R. A., Usui, T., Owen, B. A., Karcher, A., Henderson, B., Bodmer, J. L., McMurray, C. T., Carney, J. P., Petrini, J. H., and Tainer, J. A. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature, 418: 562–566, 2002.[Medline]
  48. Hirano, T. SMC protein complexes and higher-order chromosome dynamics. Curr. Opin. Cell Biol., 10: 317–322, 1998.[Medline]
  49. Kim, S. T., Xu, B., and Kastan, M. B. Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev., 16: 560–570, 2002.[Abstract/Free Full Text]
  50. Yazdi, P. T., Wang, Y., Zhao, S., Patel, N., Lee, E. Y., and Qin, J. SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes Dev., 16: 571–582, 2002.[Abstract/Free Full Text]
  51. Lim, D. S., Kim, S. T., Xu, B., Maser, R. S., Lin, J., Petrini, J. H., and Kastan, M. B. ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature, 404: 613–617, 2000.
  52. Zhao, S., Weng, Y. C., Yuan, S. S., Lin, Y. T., Hsu, H. C., Lin, S. C., Gerbino, E., Song, M. H., Zdzienicka, M. Z., Gatti, R. A., Shay, J. W., Ziv, Y., Shiloh, Y., and Lee, E. Y. Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature, 405: 473–477, 2000.
  53. Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O. A., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M. J., Redon, C., Pilch, D. R., Olaru, A., Eckhaus, M., Camerini-Otero, R. D., Tessarollo, L., Livak, F., Manova, K., Bonner, W. M., Nussenzweig, M. C., and Nussenzweig, A. Genomic instability in mice lacking histone H2AX. Science, 296: 922–927, 2002.
  54. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., and Chen, D. J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem., 276: 42462–42467, 2001.[Abstract/Free Full Text]
  55. Maser, R. S., Monsen, K. J., Nelms, B. E., and Petrini, J. H. J. hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double strand breaks. Mol. Cell. Biol., 17: 6087–6096, 1997.[Abstract]



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