Human Molecular Genetics, 2001, Vol. 10, No. 12 1287-1298
© 2001 Oxford University Press
Evidence for BLM and Topoisomerase III
interaction in genomic stability
Laboratory of Cancer Susceptibility, Department of Human Genetics and the Cell Biology Program and 1Laboratory of Molecular and Developmental Biology, Department of Human Genetics and the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA and 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
Received 24 January 2001; Revised and Accepted 2 April 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The genomic instability of persons with Bloom’s syndrome (BS) features particularly an increased number of sister-chromatid exchanges (SCEs). The primary cause of the genomic instability is mutation at BLM, which encodes a DNA helicase of the RecQ family. BLM interacts with Topoisomerase III
(Topo III), and both BLM and Topo III localize to the nuclear organelles referred to as the promyelocytic leukemia protein (PML) nuclear bodies. In this study we show, by analysis of cells that express various deletion constructs of green fluorescent protein (GFP)-tagged BLM, that the first 133 amino acids of BLM are necessary and sufficient for interaction between Topo III and BLM. The Topo III-interaction domain of BLM is not required for BLM’s localization to the PML nuclear bodies; in contrast, Topo III is recruited to the PML nuclear bodies via its interaction with BLM. Expression of a full-length BLM (amino acids 1–1417) in BS cells can correct their high SCEs to normal levels, whereas expression of a BLM fragment that lacks the Topo III interaction domain (amino acids 133–1417) results in intermediate SCE levels. The deficiency of amino acids 133–1417 in the reduction of SCEs was not explained by a defect in DNA helicase activity, because immunoprecipitated 133–1417 protein had 4-fold higher activity than GFP-BLM. The data implicate the BLM-Topo III complex in the regulation of recombination in somatic cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bloom’s syndrome (BS) is a rare, autosomal recessive disorder characterized by growth deficiency, a sun-sensitive facial erythema, hypo- and hyperpigmented skin lesions, immunodeficiency, subfertility in females and infertility in males, susceptibility to diabetes and, most importantly, a predisposition to developing cancers of all types (1). Cells derived from persons with BS exhibit excessive numbers of chromosome breaks and chromatid exchanges, including excessive numbers of sister-chromatid exchanges (SCEs) (2). Consistent with the elevation in chromosomal abnormalities, the frequency of locus-specific mutations also is increased both in vivo and in vitro (3). The hypermutability of BS cells is very likely the cause of the cancer predisposition in persons with BS; however, the molecular basis of the genomic instability in BS is poorly understood.
The gene mutated in BS, BLM, encodes a 1417 amino acid protein with a central region (residues 649–1000) containing seven motifs which are characteristic of DNA helicases (4). The region of BLM that contains these seven motifs exhibits 40–45% identity to homologous regions of other proteins in the RecQ subfamily of DNA helicases. Other family members include Escherichia coli recQ (5), Saccharomyces cerevisiae SGS1 (6), Schizosaccharomyces pombe rqh1+ (7), mammalian RECQL1 (8,9), WRN, the gene mutated in Werner syndrome (10), RECQL4, the gene mutated in some cases of Rothmund–Thomson syndrome (11,12), and RECQL5 (11). BLM has DNA-dependent ATPase activity and DNA helicase activity with a 3'
5' directionality (13). Consistent with a proposed function in recombination in somatic cells, BLM can also enter and unwind G4 tetrahelical DNA (14), Holliday junctions (15) and D-loops (16). In addition to the catalytic helicase domain, BLM and certain other RecQ helicases contain an extended (400–650 amino acids) N-terminal region which is not homologous to any other proteins. These N-terminal regions very likely determine specific interactions with protein partners which confer distinct functions to these RecQ helicases (see below).
Consistent with BS being a recessive trait, the majority of the BS mutations are null alleles (1,4). Consequently, most BS cells examined contain no detectable BLM by western blot and immunohistochemical analyses. BLM protein can be restored to BS cells by transfection of a normal BLM cDNA expression construct, and the ectopically expressed protein can correct the high-SCE phenotype (17). On the contrary, expression of BLM proteins that contain mutations in the helicase domain which disrupt its helicase activity cannot complement the high-SCE phenotype of BS cells, suggesting that BLM’s DNA helicase activity is required for its normal function in maintaining genomic stability (18).
Studies in which indirect immunofluorescence microscopy was employed have revealed that BLM is a nuclear protein which is present in both diffuse microspeckles and discrete foci (17). The discrete foci of BLM have been identified (19) as the promyelocytic leukemia protein (PML) nuclear bodies (PML-NBs, also referred to as ND10s and PODs). The PML-NB is a nuclear organelle that contains PML (20,21). Besides PML, an increasing number of other regulatory proteins, such as Sumo-1, hypophosphorylated Rb and p53, have been identified as being present in the PML-NBs, and this organelle is disrupted by several DNA tumor viruses (22), suggesting that the PML-NBs play a central regulatory function in the nucleus.
Several of the RecQ helicases have been found to interact directly with one or more topoisomerases. For example, SGS1 was first identified as a suppressor of the slow-growth phenotype of top3 null cells (6). top3 mutant cells (23) exhibit a more extreme hyper-recombination phenotype than sgs1 cells (24,25). Sgs1 interacts with both topoisomerases II (25) and III (Topo III) (6), and the N-terminal region of Sgs1 mediates these interactions (6,26–28). Interaction between RecQ helicases and topoisomerases is remarkably conserved in evolution; S.pombe rqh1+ interacts with Topo III (29,30), BLM and RECQL1 interact with Topo III (31,32), and RECQL5 interacts with Topo III and Topo IIIß (33). Topo IIIs are type IA topoisomerases which act by breaking and rejoining DNA via an enzyme–DNA intermediate in which a protein tyrosine forms an ester bond with a 5'-phosphoryl group in DNA (34). The characterized Topo IIIs are relatively inefficient at relaxation of negative supercoils; instead they prefer single-stranded DNA (ssDNA) as substrate (35–37). Consistent with the suggestion that certain RecQ helicases might provide ssDNA substrates on which Topo IIIs operate, E.coli RecQ functionally interacts with Topo III in a strand passage reaction that permits decatenation of linked, supercoiled DNA molecules (38). Like BLM, some of the human Topo III localizes to the PML-NBs, suggesting that some BLM and Topo III functions may be coordinated through this organelle (31,32).
To gain further insight into the function of BLM, we have delineated the location of the Topo III-interaction domain and investigated the role of interaction in protein localization and genomic stability in human somatic cells. We show here (i) that the first 133 amino acids of BLM mediate interaction with Topo III and are necessary for the recruitment of Topo III to the PML-NBs, and (ii) that expression of a BLM fragment that lacks this domain is only partially functional in the reduction of SCEs in BS cells, while it maintains its DNA helicase activity. These results implicate the BLM-Topo III complex in the maintenance of genomic stability.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The N-terminal region of BLM mediates interaction with Topo III
Interaction between BLM and Topo III
was detected by co-immunoprecipitation analyses with anti-BLM and anti-Topo III (31). To determine the segment of the BLM protein that interacts with Topo III, a panel of green fluorescent protein (GFP)-tagged BLM expression constructs with various deletions (Fig. 1A) were transiently transfected into the SV40-transformed BS fibroblast cell line (FCL) GM08505 (for a description of these recipient cells, see Materials and Methods), and the expressed fragments of BLM were tested for interaction with Topo III by co-immunoprecipitation analysis. In addition to being linked to GFP, the N-terminal segments of BLM were fused at the C-terminus to a 62 amino acid segment containing the nuclear localization signal (NLS) of BLM (39) in order to target the protein to the nucleus. As shown below, full-length GFP-BLM is an active DNA helicase and can correct the high SCEs of BS cells to normal levels.
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Since S.cerevisiae Sgs1 interacts with Topo III through its N-terminal region (6,26–28), we first tested constructs containing various lengths of the N-terminal region of BLM (Fig. 1B). Cells transfected with these BLM N-terminal segments fused to the NLS exhibited nuclear fluorescence (data not shown). By immunoprecipitation with anti-BLM, we found that each of the N-terminal fragments of BLM tested, including one with only the first 133 amino acids, interacted with Topo III
. Conversely, Topo III was not detected in immunoprecipitates prepared from GM08505 cells transfected with a BLM construct encoding a protein lacking the first 133 amino acids (133–1417), even though the BLM fragment was efficiently immunoprecipitated by anti-BLM. Deletion of BLM amino acid residues 131–237 or 238–340 did not impair BLM’s interaction with Topo III. Consistent with these data, the 1–133/NLS fragment, but not the 133–1417 fragment, was immunoprecipitated with anti-Topo III (Fig. 1C). The failure of the 133–1417 fragment to interact with Topo III indicated that neither the GFP tag nor the NLS of BLM themselves caused interaction with Topo III. We conclude that the first 133 amino acids of BLM are necessary and sufficient for interaction with Topo III.
BLM recruits Topo III to the PML-NBs
By indirect immunofluorescence analysis of non-mutant cells, both BLM (19) and Topo III localized to the PML-NBs in almost all cells in which BLM expression was detected; however, in BS cells in which BLM was absent, Topo III failed to localize there (Fig. 2A) (31,32). When the full-length BLM was expressed transiently by transfection into GM08505 or GM01492 cells, a nuclear distribution of BLM resulted which included cells with both diffuse and focal signals, and cells with diffuse signals only. The foci corresponded to the PML-NBs as determined by staining with anti-PML (Fig. 2B). The transient expression of GFP-BLM restored Topo III to the PML-NBs in both the SV40-transformed and untransformed BS fibroblasts, as shown by staining with anti-Topo III (Fig. 2C). On the contrary, when 133–1417 was transiently expressed in either GM08505 or GM01492 cells, although the 133–1417 fragment efficiently localized to the PML-NBs, Topo III rarely localized there (Fig. 2B and C). Similarly, Topo III localized to the PML-NBs in clones of GM08505 cells (see below) which stably expressed GFP-BLM but not in cells that expressed 133–1417 (Fig. 2D). In GM08505 cells that transiently expressed a GFP-BLM fragment deleted for amino acids 131–237, both BLM and Topo III failed to localize to the PML-NBs (Fig. 2E); however, the 
131–237 fragment and Topo III were capable of interaction (Fig. 1). Consistent with these findings, transient expression in SV40-transformed normal fibroblasts of the 1–133/NLS fragment, which failed to localize to the PML-NBs, resulted in the inhibition of transit of Topo III into the PML-NBs (Fig. 2F), presumably by competing with the endogenous BLM for binding to Topo III. As a control, we showed this inhibition was not detected in cells transiently expressing GFP. These results indicated (i) that the region of BLM that mediates localization to the PML-NBs is separate and independent of the Topo III-interaction domain and (ii) that BLM and Topo III interaction can occur both inside and outside of the PML-NBs. We conclude that BLM recruits Topo III to the PML-NBs.
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133–1417 is deficient in the correction of the high-SCE phenotype of BS cells
We have shown that the high-SCE phenotype of BS cells can be corrected by transfection of a normal BLM cDNA into them (17). To investigate the role of BLM-Topo III
interaction in correcting SCEs, we compared the SCE frequencies of GM08505 cells that stably expressed 133–1417 with those of cells that stably expressed GFP-BLM. For SCE analysis, we selected two clones that expressed each of these constructs at elevated levels by GFP fluorescence and western blot analysis, because our earlier work had shown that normal to lower levels of BLM expression in GM08505 cells resulted in less SCE correction (17). Two clones that expressed GFP alone were also tested as controls.
The levels of protein expressed in each of two clones transfected with GFP-BLM and with 133–1417 were roughly equal as determined by probing immunoblots with anti-GFP. They were 
10-fold greater than the levels of expression of endogenous BLM in the SV40-transformed normal fibroblast GM00637 as determined by probing with anti-BLM (Fig. 3).
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To test the function of the expressed BLM proteins in genomic stability, we counted the numbers of SCEs in metaphases from each cell line (Fig. 4). Similar to our previous observations, in the two cells lines that stably expressed GFP-BLM the mean numbers of SCEs per 46 chromosomes were 11.6 ± 2.8 and 13.7 ± 4.4. In contrast, in the two cell lines that expressed 133–1417, the mean numbers of SCEs per 46 chromosomes were 36 ± 12.8 and 53.8 ± 23. The differences between the distribution of SCEs in cells that expressed GFP-BLM and 133–1417 were statistically significant (P < 0.001). In 133–1417 clone 2, five metaphases with over 80 SCEs per 46 chromosomes were detected (Fig. 4), possibly representing a subpopulation of cells with lower levels of 133–1417 expression. In the two clones of GM08505 that expressed GFP, the mean numbers of SCEs per 46 chromosomes were 77.6 ± 10.6 and 83.6 ± 12.7, similar to that found in untransfected cells (75.1 ± 10.1). The distributions of SCEs in GFP-transfected and untransformed GM08505 cells was also significantly different from the distribution of SCEs in the two clones that expressed 133–1417 (P < 0.001). These results showed that, compared with full-length BLM, a BLM lacking the Topo III
-interaction domain is deficient in correction of the high-SCE phenotype of BS cells.
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133–1417 has elevated helicase activity
To test whether the deletion of amino acids 1–133 of BLM impairs its helicase activity, thereby explaining the deficiency of 133–1417 in correction of the high SCEs of GM08505 cells, we compared the helicase activities of GFP-BLM and 133–1417 by the strand displacement assay. GFP-BLM and 133–1417 proteins were partially purified by immunoprecipitation from one clone each (Fig. 5) of the two clones that stably expressed them. Both GFP-BLM and 133–1417 exhibited detectable strand-displacement activities (Fig. 5A), and the amounts of displaced substrate were proportional to the amounts of precipitated proteins added to the assay as determined by probing with anti-GFP (Fig. 5B). No helicase activity was detected in the immunoprecipitates prepared from untransfected GM08505 cells, indicating that contaminating DNA helicases were not detected in the assay. Quantitative western blot analysis was performed to measure the amounts of BLM present in the immunoprecipitates prepared with anti-BLM (Fig. 5B and C), and the relative helicase activities of the GFP-BLM and 133–1417 proteins were calculated (Fig. 5D). The activity of 133–1417 was
4-fold higher than GFP-BLM (Fig. 5D). As in the transient transfection assay, we confirmed that Topo III was present in immunoprecipitates prepared from BS cells that expressed GFP-BLM but not in cells that expressed 133–1417 (Fig. 5E). These data demonstrated that the deficiency of 133–1417 in correction of the high SCEs in BS cells was not explained by reduced helicase activity of the 133–1417 protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Topo III
interaction domainWe have investigated here the role of interaction between BLM and Topo III
in the maintenance of genomic stability. By a co-immunoprecipitation assay of extracts of transiently transfected BS cells, we found that the first 133 amino acids of BLM, when linked to GFP and the NLS of BLM, are necessary and sufficient for interaction with Topo III. Wu et al. (32) have reported that a fragment of BLM consisting of amino acids 1–212 interacted with purified Topo III by far-western analysis whereas a fragment consisting of 1–142 did not, and they concluded that BLM residues 142–212 are important for interaction with Topo III. In addition, the C-terminal 150 amino acids of BLM were found to interact with Topo III. We can explain the inconsistencies between the two sets of results in the following way. Firstly, we have used transfection and co-immunoprecipitation analysis (Fig. 1), which tests the interaction between the two proteins under conditions that are closer to the physiological state than far-western analysis. Secondly, partial trypsin digestion analysis of a purified N-terminal 431 amino acid fragment of BLM (40) has suggested that amino acids 1–136 and 137–204 form independently folding protein segments (unpublished data). We have found that a BLM fragment 1–131 expressed in and purified from E.coli forms an insoluble aggregate, whereas the fragments 136–208 and 1–208 are both soluble (data not shown). These results suggest that the BLM 1–131 fragment folds poorly on its own and that the region from 137 to 204 stabilizes folding of the 1–136 segment. Binding of Topo III probably depends on normal folding of the 1–136 domain, and in our experiments GFP may have stabilized that folding; the glutathione S-transferase protein used by Wu et al. (32) may have not. Furthermore, we found here that the deletion of residues 131–237 did not impair the interaction between BLM and Topo III (Fig. 1), indicating that this region of BLM is not required for interaction in our assays. Because the GFP-BLM 133–1417 fragment did not interact with Topo III, neither the GFP nor the NLS moieties themselves mediated interaction. We conclude therefore that BLM residues 1–133 provide the residues critical for interaction with Topo III. Similar to our results with BLM, interaction between S.cerevisiae Sgs1 and Topo III is also mediated by the N-terminal 170 amino acids of Sgs1 (26–28), and expression of an Sgs1 that is deleted for the interaction domain is deficient in complementation of the hyper-recombination phenotype of sgs1 null cells (41).
Evidence for a role of the PML-NBs in maintenance of genomic stability
Both BLM and Topo III have been localized to the PML-NBs (19,31). Because deletion of the Topo III-interaction domain of BLM did not affect its localization to the PML-NBs (Fig. 2), the localization of BLM to the PML-NBs is independent of its interaction with Topo III. Conversely, Topo III is dependent on BLM to localize to the PML-NBs, because transfection of GFP-BLM restored Topo III to the PML-NBs but transfection of the 133–1417 construct which lacks the Topo III-interaction domain did not (Fig. 2). Lastly, expression of the GFP-BLM fragment 1–133/NLS resulted in inhibition of recruitment of Topo III to the PML-NBs, presumably by competing with endogenous BLM for binding to Topo III. These data indicate that BLM recruits Topo III to the PML-NBs.
Several lines of evidence suggest that the PML-NBs play an important role in controlling genomic stability. Many proteins involved in DNA repair or recombination have been detected there under various conditions (42–45). For example, Bischof et al. (46) have shown that a significant fraction of the foci induced after 
-ray treatment contain BLM, RPA, Rad51 and PML, and in BrdU-labeled cells ssDNA can be detected in these irradiation-induced foci. In mouse PML–/– cells, in which the PML-NBs are disrupted, the normal nuclear distribution of BLM is disrupted and the numbers of SCEs in these cells are increased 2-fold compared with normal cells (19). Finally, the 133–237 protein, when stably expressed in BS cells, failed to localize to the PML-NBs (Fig. 2E) and these cells have intermediate numbers of SCEs similar to those obtained here with 133–1417 (unpublished data). Because the SCEs in PML knockout mice are not elevated to the same extent as in BS cells, there is probably a function of BLM in SCE suppression that is independent of the PML-NBs.
BLM-Topo III interaction and SCE correction
The observation that BLM and Topo III physically interact suggested that the two proteins, as a complex, perform some function in genomic stability; hence, we expressed 133–1417 in BS cells to determine whether the absence of interaction limited the protein’s ability to correct the high SCEs. In the two cell lines that stably expressed 133–1417, the average numbers of SCEs were 38 and 53.8, whereas the numbers of SCEs in cells expressing full-length GFP-BLM were 11.6 and 13.7. The expression levels of 133–1417 and GFP-BLM were similar and 133–1417 is no less active as a helicase compared with GFP-BLM. Consequently, the fact that the numbers of SCEs observed in cells that expressed 133–1417 were intermediate suggests strongly that some part of the function of the BLM-Topo III complex in SCE correction is dependent on the interaction between these two enzymes, although we cannot rule out the possibility that some other function of the 1–133 domain is important as well. Perhaps the function of each protein acting independently and sequentially is quantitatively less efficient in SCE reduction than the two proteins acting together in a coordinated manner.
Absence of the BLM protein from a proliferating cell results in a striking genomic instability in which elevated SCEs are characteristic. The only other mammalian mutation known that increases SCEs as dramatically as in BS is in the ligase III-associated XRCC1 gene product (47), which functions in a pathway of oxidative DNA-damage repair. Certain DNA-damaging agents can induce SCEs potently, possibly by stalling the replication fork at the site of the lesion and forcing repair and restart by a recombination event between the sister chromatids (48–50). Consistent with a function for BLM in replication, BLM protein levels increase from a low at the G1/S boundary to a high by late S phase (51–54), and in BS cells, the rate of fork progression is retarded and abnormal replication intermediates are detected (55,56). Thus, the function of BLM in SCE reduction could be to prevent the formation of lesions that inhibit replication fork progression or to interact directly with components of the replication or recombination machinery and to modulate the response to an SCE-inducing lesion. Whichever is the case, our results support the hypothesis that interaction between BLM and Topo III is needed for the efficient reduction of SCEs and, by extension, for the appropriate regulation of recombination (57,58).
Manifested by elevated SCEs, the genomic instability of BS cells containing a BLM protein that fails to interact with Topo III implicates Topo III directly in DNA transactions which control recombination in mammalian somatic cells. BLM and Topo III are part of a complex that presumably coordinates the individual activities of the two proteins on their target DNA substrates. Identification of these substrates in vivo and characterization of the other proteins involved in resolving them will be key to understanding the molecular pathogenesis of BS cells and to elucidating the role of this complex in the control of recombination in normal cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell lines
The SV40-transformed FCLs GM08505 and GM00637 were obtained from the Coriell Institute for Medical Research via Dr James German (Cornell University Medical College). GM08505 is a BS cell line which exhibits the high-SCE phenotype; it was derived from a skin biopsy sample of an Ashkenazi Jewish woman homozygous for the blmAsh mutation (17). Homozygosity for blmAsh results in an 80% reduction in the steady-state levels of BLM mRNA and premature translation termination at codon 740 of BLM. The control GM00637 cell line was established from a skin sample of a normal adult. The untransformed fibroblast GM01492 (obtained from the Coriell Institute) is a BS cell line that exhibits high SCEs and is also homozygous for blmAsh. HeLa cells were obtained from M. Sadelain (Memorial Sloan-Kettering Cancer Center) and the untransformed normal FCL (CRL 1475) was obtained from R. Chaganti (Memorial Sloan-Kettering Cancer Center). GM08505, GM00637 and HeLa cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (Life Technologies) and 200 U/ml Penicillin-Streptomycin (Life Technologies). CRL1475 and GM01492 were cultured in DMEM with 15% (v/v) fetal bovine serum, 2x non-essential amino acid (Life Technologies), 2 µM glutamine (Life Technologies), 2x vitamins (Life Technologies) and 200 U/ml Penicillin-Streptomycin.
Antibodies
The rabbit anti-BLM was raised against the first 431 amino acids of BLM (40); antibodies were affinity-purified by binding to and elution from antigen attached to cyanogen bromide-activated sepharose. These antibodies have been used in a number of different molecular and cellular studies of BLM (17–19,45,53,54). The chicken anti-Topo III (HM1002) was generated as described by Johnson et al. (31). The GFP mouse monoclonal antibody was purchased from Clontech. The mouse monoclonal anti-PML antibody (PG-M3) was purchased from Santa Cruz.
Plasmids and transfection
The normal GFP-BLM construct was generated by subcloning a BLM cDNA in-frame into the EGFP-C1 vector (Clontech). Individual N-terminal segments of BLM were generated by PCR amplification of the BLM cDNA using Pfu polymerase (Clontech) with oligonucleotide primers corresponding to various sites. To generate the NLS of BLM, a segment of BLM corresponding to amino acids 1317–1378 was amplified by PCR with oligonucleotides 5'-CCGCTCGAGGAAATACCCGTATCTTC-3' and 5'-CCGGATCCAATGATGCTGGAG-3'. The various N-terminal segments and the NLS fragment were then ligated together, subcloned into the EGFP-C1 vector, and DNA sequencing was performed to verify each construct. To produce 133–1417, the GFP-BLM construct was digested with SmaI and SacII to generate a BLM fragment corresponding to amino acids 133–1417. This fragment was then subcloned into the ScaI and SacII sites of the EGFP-C3 vector. To generate the internal deletion in 131–237, oligonucleotides 5'-GTAAAGAAATCCATCGATGATGGCCCCATTGC-3' and 5'-CATCATCGATGGATTTCTTTACAGTTGGTG-3', each joining non-contiguous sequences of BLM, were paired with oligonucleotides 5'-GGAAGCGGCCGCAGTCATTTATTTATTGAAGCAGTATG-3' and 5'-GATCACATGGTCCTGCTGGAGTTC-3' in PCR using the GFP-BLM construct as a template to amplify segments corresponding to BLM residues 1–133 and 237–542. These two PCR fragments were subsequently mixed and joined by PCR with two nested primers (5'-GATCACATGGTCCTGCTGGAGTTC-3' and 5'-TTCCTAGGGTGGTAGTCAGTAA-3'). The secondary PCR fragment was then cut with Xhol and EcoR1 and cloned into the corresponding region in GFP-BLM. 238–340 was made similarly except that the two joining primers were 5'-GATGTGATTTGCGATCTTTTGTCAAAACCTGAG-3' and 5'-CAAAAGATCGCAAATCACATCGCTGCTTAAC-3'.
Transfection of the GFP-BLM constructs into BS cells was carried out using Lipofectamine (Life Technologies). For transient transfection and immunoprecipitation, 4 µg of each plasmid was combined with 30 µl of Lipofectamine reagent and incubated with GM08505 cells at 60% confluency in a 100 mm plate. For transient transfection and immunofluorescence, 1 µg of each plasmid was transfected into GM08505 or GM01492 in a 6-well plate with a glass coverslip at the bottom. Transfection efficiency was monitored by immunofluorescence microscopy of living cells, scoring green cells as expressing the GFP-BLM transgenes. Immunofluorescence and immunoprecipitation were performed 24 h after transfection. To select clones that stably expressed GFP, GFP-BLM and 133–1417 proteins, geneticin (Life Technologies) was added to the medium at 0.2 mg/ml 48 h after transfection and clones were picked into fresh wells after 3 weeks. After evaluation of protein levels by western blot analysis, two clones expressing high levels of each BLM construct were selected for further analyses.
Immunoprecipitation
Twenty-four hours after transfection of GFP-BLM constructs, 2 x 106 cells were directly lysed in the tissue culture dish with 1 ml of NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40 and 50 mM Tris–HCl pH 8.0) at 4°C for 1 h. Extracts were pre-cleaned by centrifugation and then mixed with antibodies. The anti-BLM was added at 1:100 dilution and the chicken anti-Topo III antibody (HM1002) was added at 1:300 dilution. After immune complexes were allowed to form, 20 µl of protein A– or G–Sepharose (Sigma) were added, and samples were incubated with gentle rocking at 4°C for another hour. Protein A was used to capture the rabbit anti-BLM and protein G was used to capture the chicken anti-Topo III. The immune complexes were pelleted by centrifugation, washed three times in 1 ml of lysis buffer and then boiled in 20 µl of 1x SDS sample buffer (50 mM Tris–HCl pH 6.8, 100 mM DTT, 2% SDS, 0.2% bromophenol blue and 20% glycerol). For western analysis, the samples (10 µl) were separated on a 6% SDS–PAGE and blotted by standard methods. The immunoblots were then blocked in PBS containing 3% Tween-20 and 10% non-fat dry milk (blocking buffer) for 1 h and then probed with anti-Topo III HM1002 at 1:5000, anti-BLM at 1:500 or anti-GFP at 1:500 diluted in blocking buffer. Horseradish peroxidase-conjugated goat IgGs (Vector) diluted 1:5000 in blocking buffer were used as the secondary antibodies and chemiluminescent detection was performed using the ECL kit (Amersham).
Indirect immunofluorescence
For immunofluorescence analyses, GM00637, GM08505, CRL1475 or GM01492 fibroblasts were grown on glass coverslips in a 6-well plate to 80% confluence. Cells were transfected with GFP-BLM constructs as described above. Cells were fixed and permeabilized in 2% paraformaldehyde, 0.1% Triton-X100 at 4°C for 20 min. After washing three times in PBS, cells were blocked in PBS supplemented with 10% (v/v) bovine serum albumin at room temperature for 1 h. After blocking, antibodies (1:200 for anti-PML, 1:500 for anti-Topo III and 1:200 for anti-BLM) diluted in blocking buffer were applied to the coverslips and incubated with the cells at room temperature for 1 h. Coverslips were then washed three times again and incubated with Texas red- or fluorescein isothiocyanate (FITC)-secondary antibodies (Vector) diluted in blocking buffer with 1 ng/ml DAPI at room temperature for another hour. After washing, the coverslips were mounted on a glass slide in Vectashield mounting medium (Vector). Slides were viewed at 100x magnification with an Olympus fluorescence microscope mounted with a Sony charge coupled device (CCD) camera. Cells were evaluated for the presence or absence of foci, which were defined as intensely fluorescent signals that were 0.2–1 µm in size and circular in shape. Cells with and without foci were tabulated (a minimum of 100 randomly encountered cells). When two foci overlapped in position and the color of the fluorescence on viewing with the triple band pass filter was yellow, then the foci were said to co-localize. The percentage co-localization [(2 x no. co-localized foci/no. green foci + no. red foci) x 100] was calculated on a minimum of nine randomly encountered cells in which foci were detected (over 100 foci were counted). Images were captured, pseudocolored and merged in Abode Photoshop.
Quantitative western blot analysis
For quantitative western analysis of BLM in whole cell extracts, 2 x 106 cells of each cell line that stably expressed a BLM construct were directly lysed on the plates in 500 µl of 1x SDS sample buffer without bromophenol blue. Extracts were passed through a 26-gauge needle to shear the DNA and they were boiled for 5 min. A volume of extract corresponding to 20 000 cells was loaded on a 6% SDS–polyacrylamide gel, and the gel was blotted and probed as described above. For analysis of the immunoprecipitates used in the helicase assay (see below), amounts of GM08505 cell lysates corresponding to 20 000 cells were mixed with aliquots of immunoprecipitation products to ensure even gel-transfer of BLM during western blotting. To measure the molar amount of GFP-BLM, a series of dilutions of the immunoprecipitation products was mixed with a series of dilutions of BLM N431 (40). BLM N431 contains the first 431 amino acids of BLM fused with a hexahistidine tag, expressed in and purified from E.coli. Because BLM N431 was used to generate the anti-BLM, it contains the same epitopes for the anti-BLM as the full-length BLM. The exact amount of purified BLM N431 was measured by the Bradford method (Biorad) and SDS–PAGE using a series of dilutions of BSA as standard.
SCE analysis
The differential staining of sister chromatids in metaphases prepared for GM08505, GM00637 and clones of GM08505 cells that stably expressed different GFP-BLM constructs or GFP alone were performed as described by Ellis et al. (17). Pictures of intact metaphases taken at 100x magnification were captured with the CCD camera and SCEs were counted by analysis of these pictures. Statistical analyses were performed using the Student’s two-tailed t-test and the significance level was set at 0.0014, which is the value obtained by adjusting the 0.05 level for the number of tests performed (the Bonferoni correction; in this case dividing 0.05 by 27).
Oligonucleotide displacement assay
To make the substrate for the standard helicase assay, a 90 bp oligonucleotide (13) was annealed to M13mp18 ssDNA. The 3' end of the annealed oligonucleotide was labeled by incorporation of [32P]-dCTP using the Klenow fragment of DNA polymerase I (New England Biolabs) at 25° for 20 min. The labeled substrates were separated from unincorporated nucleotide and unannealed oligonucleotide by centrifugation through a spin column (Biorad spin-400) according to the manufacturer’s instructions. Helicase assays were then performed on immunoprecipitated BLM as follows: cell lysates were first prepared from 15–30 x 106 untransfected or stably transfected GM08505 cells in 6 ml of lysis buffer (20 mM Tris–HCl pH 8.0, 0.4 M NaCl, 1mM EDTA, 0.5 mM DTT, 0.5% NP-40 and 25% glycerol). Immune precipitates were prepared as described above. After washing, the immune complexes were re-suspended in 200 µl of 1x helicase buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 5 mM ATP, 100 µg/ml BSA and 50 mM NaCl). Aliquots of resuspended immune complexes were immediately mixed with 5 ng of substrate in 40 µl of 1x helicase buffer, and the reaction was incubated at 37°C for 30 min. The reaction was stopped by adding 5 µl of stop solution (30% glycerol, 150 mM EDTA and 2% SDS) and analyzed by electrophoresis of the reaction products through a 12% non-denaturing polyacrylamide gel. As controls, untreated substrate and substrate that was heated at 100°C for 5 min before loading on the gel were analyzed. After electrophoresis, the gel was dried and exposed in a phosphoimager system (Biorad) for quantification of the signals.
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ACKNOWLEDGEMENTS |
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This research was supported by Memorial Sloan-Kettering Cancer Institute and the May and Samuel Rudin Family Foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Memorial Sloan-Kettering Cancer Center, Box 124, 1275 York Avenue, New York, NY 10021, USA; Tel: +1 212 639 7183; Fax: +1 212 717 3571; Email: n-ellis@ski.mskcc.org
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REFERENCES |
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A. Franchitto and P. Pichierri Bloom's syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest J. Cell Biol., April 1, 2002; 157(1): 19 - 30. [Abstract] [Full Text] [PDF]
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