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Recruitment of HRDC domain of WRN and BLM to the sites of DNA damage induced by mitomycin C and methyl methanesulfonate
Saheli Samanta and Parimal Karmakar1
Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India
The HRDC (helicase and RNase D C-terminal) domain at the C-terminal of WRNp (Werner protein) (1150–1229 amino acids) and BLMp (Bloom protein) (1212–1292 amino acids) recognize laser microirradiation-induced DNA dsbs (double-strand breaks). However, their role in the recognition of DNA damage other than dsbs has not been reported. In this work, we show that HRDC domain of both the proteins can be recruited to the DNA damage induced by MMS (methyl methanesulfonate) and MMC (methyl mitomycin C). GFP (green fluorescent protein)-tagged HRDC domain produces distinct foci-like respective wild-types after DNA damage induced by the said agents and co-localize with γ-H2AX. However, in time course experiment, we observed that the foci of HRDC domain exist after 24 h of removal of the damaging agents, while the foci of full-length protein disappear completely. This indicates that the repair events are not completed by the presence of protein corresponding to only the HRDC domain. Consequently, cells overexpressing the HRDC domain fail to survive after DNA damage, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Moreover, 24 h after removal of damaging agents, the extent of DNA damage is greater in cells overexpressing HRDC domain compared with corresponding wild-types, as observed by comet assay. Thus, our observations suggest that HRDC domain of both WRN and BLM can also recognize different types of DNA damages, but for the successful repair they fail to respond to subsequent repair events.
Key words: Bloom, comet assay, HRDC domain, MMC, MMS, Werner
Abbreviations: BER, base excision repair, BLMp, Bloom protein, dsb, double-strand break, EGFP, enhanced green fluorescent protein, FBS, fetal bovine serum, GFP, green fluorescent protein, HR, homologous recombination, HRDC, helicase and RNase D C-terminal, LP-BER, long-patch BER, MMC, methyl mitomycin C, MMS, methyl methanesulfonate, MTT, 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl-2H-tetrazolium bromide, PCNA, proliferating-cell nuclear antigen, RQC, C-terminal domain of RecQ, WRNp, Werner protein
1To whom correspondence should be addressed (email firstname.lastname@example.org).
The RecQ helicases, one of the most important families of DNA helicases, highly conserved from Escherichia coli to humans, engage in different DNA metabolisms and are required for the maintenance of genome integrity. In humans, there are 5 RecQ homologues, and mutations in 3 of these genes BLM (Bloom), WRN (Werner), Rothmund–Thomson (RTS/RECQ4) are associated with Bloom, Werner and Rothmund-Thomson syndromes respectively. These 3 are associated with hereditary disorders manifesting genomic instability, cancer predisposition and/or premature aging (Bernstein et al., 2010). Among RecQ helicases, a similar overall structural organization is associated with WRN and BLM, where the helicase domain, containing a DEAH box, functions to unwind DNA in an ATP- and Mg2+-dependent manner (Bernstein et al., 2010). The RQC (C-terminal domain of RecQ) is responsible for protein–protein interactions (Kobbe and Bohr, 2002; Hickson, 2003; Bernstein et al., 2010; Vindigni et al., 2010). In addition to the helicase domain and RQC, they also possess acidic patches and NLS (nuclear localization signal) in their C-terminus (Kaneko et al., 1997; Matsumoto et al., 1997; Hickson, 2003; Bernstein et al., 2010). With an exception WRN is the only member of RecQ helicases that possesses a 3′–5′ exonuclease domain (Bukowy et al., 2008). In humans, only WRN and BLM possess the HRDC (helicase and RNase D C-terminal) domain (80 amino acids) at their C-terminus (Bernstein et al., 2010). In vitro helicase activity of BLM and WRN or the exonuclease activity of WRN has been well studied (Yang et al., 2002; Opresko et al., 2003). The proteins corresponding to BLM and WRN, BLMp and WRNp (Werner protein) interact with various DNA substrates which arise during different DNA metabolic activities, such as restoration of replication fork migration, recombination and/or repair. Although in vitro studies indicate their active participation in DNA repair, their activities in vivo remain unclear. Several DNA repair proteins change their subnuclear localization after DNA damage and are concentrated at the damaged sites called nuclear foci (Maser et al., 1997; Sakamoto et al., 2001). These foci are the sites of DNA damage and DNA repair proteins accumulate at these sites after DNA damage. Restoration of their localization indicates the completion of DNA repair events (Karmakar and Bohr, 2005).
Both WRNp and BLMp form distinct foci after various kinds of DNA damage (Bischof et al., 2001; Sakamoto et al., 2001). Experiments on live cells showed that after laser microirradiation-induced DNA dsbs (double-strand breaks), protein corresponding to HRDC domain of WRN and BLM is recruited to the damage sites (Lan et al., 2005; Karmakar et al., 2006). Also, from in vitro studies, it is proposed that purified proteins of HRDC domain of BLM is required for the resolution of Holliday junction (Wu and Hickson, 2005; Sharma et al., 2007). Thus, HRDC domain of both the proteins is essential for the repair of DNA dsbs at least at the initial recognition step. Given that both WRNp and BLMp form distinct foci after DNA damage induced by a variety of agents, it is still unknown which domain of WRN and BLM accumulates at the damage sites other than DNA dsbs. Active role of WRNp in BER (base excision repair) has already been reported (Karmakar and Bohr, 2005; Harrigan et al., 2006; Muftuoglu et al., 2008). Both WRNp interact with several proteins involved in BER and some of them can modulate its catalytic function in vitro. Among the different domains present, HRDC domain of WRN and BLM may play additional role as they are absent in other human RecQ family member such as RecQ1, RecQ 4 and all isoforms of RecQ5.
In the present study, we have examined the potential of HRDC domain in recognizing DNA damages other than DNA dsbs. BLM-HRDC and WRN-HRDC co-localized with γ-H2AX after DNA damage induced by a DNA cross-linker, MMC (methyl mitomycin C) or an alkylating agent MMS (methyl methanesulfonate). We have also observed that cells overexpressing HRDC domain only failed to survive after DNA damage induced by MMC and MMS. Incomplete DNA repair in cells overexpressing HRDC domain is largely attributed to the lack of survival of these cells. Thus, the HRDC domain of both WRN and BLM is responsible for damage recognition, but other domains of both the proteins are necessary for successful DNA repair.
2. Materials and methods
pEGFPC1 vector (Clontech) encodes the full-length human wild-type WRN, and BLM and BLM-HRDC were provided by Professor T. Emomoto (Sendai, Japan). Plasmid containing WRN was used as a template to amplify by PCR the fragment containing the HRDC domain using an upstream primer 5′-tactcgagggcaaattggtagaagctag-3′ and a downstream primer 5′-atggatccttaactaaaaagacctcccc-3′ at BamI/Xho I sites, using pfu (plaque-forming units) polymerase (Stratagene).
2.2. Cell culture and transfection
HeLa cells was purchased from National Centre for Cell Science, grown in monolayer in DMEM (Dulbecco's modified Eagle medium; Sigma) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 2 mM l-glutamine, 100 units/ml streptomycin and 100 units/ml penicillin and maintained at 37°C, 5% CO2 and 95% air with RH (relative humidity). Transient transfections of pEGFPC1-WRN, pEGFPC1-WRN-HRDC, pEGFPC1-BLM, pEGFPC1-BLM-HRDC and empty vector (pEGFPC1) were performed with Transpass D1 (New England Biolabs). Cells were seeded 24 h prior to transfection in coverslips at 3–2×104 cells/ml.
2.3. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay
MTT assay was used in the cells either mock treated or treated with DNA damaging agents, MMC and/or MMS. After 24 h of transfection, the cells were treated with MMC (0.5 μg/ml) for 16 h and MMS (1 mM) for 1 h, washed once with PBS and incubated in fresh medium for a further 24 h. Cells were washed once with PBS and incubated in Phenol Red-free medium with MTT (450 μg/ml) for 4 h at 37°C. The formazan crystals were dissolved in an MTT solubilization buffer and absorbances measured with a UV-visible spectrophotometer (Hitachi) at 570 nm (Maanen et al., 1988).
After 24 h of transfection, cells were treated with MMC for 16 h (0.5 μg/ml) or MMS (1 mM) for 1 h. Cells just after treatment (0 h) and after 24 h of withdrawal of damaging agents were collected for immunofluorescence analysis. Cells were washed with PBS, fixed with freshly prepared 4% PFA (paraformaldehyde) for 10 min at room temperature. They were washed thrice with PBS and permeabilized with 0.25% Triton X-100 in ice for 10 min before being washed with cold PBS thrice. They were incubated with 5% FBS in PBS for 1 h at room temperature. Proteins were detected immunologically by incubating the coverslips with the anti-γ-H2AX mouse monoclonal antibody (Upstate) for 16 h at 4°C. After washing thrice (10 min each) with PBS containing 0.05% Tween and 0.5% FBS, the coverslips were incubated simultaneously with secondary antibodies conjugated with fluorescence dye (anti-rabbit and anti-mouse 568, Alexa Fluor®, Molecular Probes) for 1 h at room temperature. After washing thrice (10 min each), the coverslips were mounted on Vectashield (Vector Laboratories) and viewed under a fluorescence microscope (Leica) using Leica FW4000 software. The images were processed using Adobe Photoshop software.
2.5. Single-cell gel electrophoresis study
Comet assay slides were prepared using the protocol of Szeto et al. (2005). Briefly, 200 μl of normal-melting-point agarose was pipetted on to frosted slides and spread by the lowering of coverslips (22×50 mm) to ensure an even agarose spread, and left briefly to solidify. Coverslips were removed. Cells were re-suspended in 100 μl of low-melting-point agarose that was in turn pipetted on to the normal-melting-point agarose layer, spread with coverslips and left briefly to solidify. Then 50 μl of trypsin solution [0.25% trypsin and 1 mM EDTA in HBSS (Hanks balanced salt solution)] was added to the slides and left for 30 min at 37°C. Thereafter the slides were washed with ice-cold PBS, and 50 μl of proteinase K (Sigma Chemicals) solution (1 mg/ml of PBS) was applied to the slides and left for 1 h at 4°C. Slide were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris/HCl, pH 10, 1.0% Triton X-100) and incubated for 1 h at 4°C. These slides were equilibrated in an electrophoresis buffer (0.01 M NaOH and 1 mM EDTA, pH 9.1) for 20 min at 4°C before being electrophoresed at 12 V (constant voltage) for 20 min at room temperature. The slides were drained, flooded slowly with three changes of neutralization buffer (0.4 M Tris, pH 7.4) before being stained with 40 μl of 20 μg/ml ethidium bromide (Sigma Chemicals) and examined immediately under a fluorescence microscope (Leica DM 4000B). A total of ∼50 cells per slide were analysed for comet parameters using an image analysis system (Komet version 5.5, Kinetic Imaging Ltd) (Szeto et al., 2005).
2.6. Statistical analysis
A Student's t test was used to calculate the statistical significance of changes. In all cases, differences are significant for P<0.05 unless stated otherwise (*P<0.05, **P<0.001 and ***P<0.0001). Data analyses were performed using the Prism software (GraphPad).
3.1. Response of cells transfected with full-length WRN or BLM or their respective HRDC domain after treatment with MMC and MMS
In order to investigate the role of HRDC domains of RecQ family members, WRN and BLM in DNA damage response other than dsbs, we transiently overexpressed HeLa cells with one of the following EGFP (enhanced green fluorescent protein) constructs separately: full-length WRN, BLM, their respective HRDC domains or the empty vector. After transfection, the expression levels of the proteins were determined by immunoblotting whole cell lysate with antibody against GFP (green fluorescent protein) (Supplementary Figure S1 available at http://www.cellbiolint.org/cbi/036/cbi0360873add.htm). After transfecting these EGFP-constructs in HeLa cells, cell survival was quantified after recovery from DNA damage induced by MMS and MMC. Cells were treated with different concentrations of DNA damaging agents and allowed to recover in fresh medium for 24 h after removal of damaging agents, before being assayed with MTT. The cells overexpressing any of the HRDC domains are sensitive to MMC and they fail to recover after 24 h of damage compared with respective full-length protein (Figure 1A). Similarly cells overexpressing HRDC domain of either gene survive much less compared with respective full-length protein when treated with MMS (Figure 1B).
3.2. Accumulation of WRN HRDC and BLM HRDC at sites of DNA damage induced by MMC and MMS
To verify whether both the damaging agents can induce sufficient DNA damage, a control experiment was carried out. HeLa cells were treated with MMC and MMS with respective LD50 doses (0.5 μg/ml MMC and 1 mM MMS) and immunostained with PCNA (proliferating-cell nuclear antigen), a protein involved in DNA damage response pathways. Distinct PCNA foci for these doses were seen (Supplementary Figure S2 available at http://www.cellbiolint.org/cbi/036/cbi0360873add.htm), indicating that these doses caused DNA damage. The question was raised whether the HRDC domain could recognize the damage induced by the DNA damaging agents. Overexpressed cells were incubated with the damaging agents (0.5 μg/ml MMC, 1 mM MMS at LD50 concentrations obtained from Figure 1) after transfection with GFP-tagged construct, for 16 h with MMC and 1 h with MMS (considered as 0 h in Figure 1), and allowed to grow in fresh medium in the absence of damaging agents (considered as 24 h in Figure 1). As seen in the upper panel of Figure 2(A) after DNA was damaged with MMC, WRNp overexpressing cells show distinct nuclear foci that co-localize with γ-H2AX. After 24 h of recovery, most of WRNp was restored to the nucleolus as seen in untreated control cells (first row of Figure 2A). A similar distribution was also found for BLMp (Figure 2B, upper panel) after MMC treatment. Their HRDC domain (lower panel of Figures 2A and 2B) are also able to produce distinct nuclear foci after the treatment with this damaging agent, but after 24 h of recovery they failed or partially failed to be restored. Similarly, full-length WRNp and BLMp recruited to sites of γ-H2AX after the treatment with MMS and were restored to their position after 24 h recovery (Figures 2C and 2D respectively). However, their HRDC domain failed to restore during recovery from MMS induced damage, although they co-localize with γ-H2AX after DNA damage (lower panel of Figures 2C and 2D). We also estimated the number of cells having foci in all the cases. In Table 1, the percentage of γ-H2AX foci positive cells were estimated at 0 and 24 h of recovery. More γ-H2 AX foci were associated with the cells transfected with HRDC domain of WRN or BLM after 24 h of recovery (P<0.02 for WRNp and P<0.0001 for BLMp in MMC induced damage, with P<0.02 for WRNp and P<0.0001 for BLM in MMS induced damage), although the amount of foci positive cells at 0 h remained the same as the respective full-length protein. The percentage of foci produced by GFP-tagged proteins was also estimated (Table 2). Here also the amount of GFP foci for HRDC protein remained high compared with the respective full-length proteins after 24 h of recovery (P<0.011 for WRNp, P<0.0001 for BLM after MMC treatment, P<0.0005 for WRNp and P<0.01 for BLMp after MMS treatment). These experiments, along with our data from Figures 1(A) and 1(B), show that the HRDC domain can recognize the damage induced by MMS or MMC, but that subsequent DNA repair events are impaired. To ensure that HRDC domains of both WRN and BLM cannot complete the repair in damaged cells, we have determined that neither of endogenous WRN nor BLM returned to the damaged sites in HeLa cells after 24 h of recovery from the damage induced by these agents (Supplementary Figure S3 available at http://www.cellbiolint.org/cbi/036/cbi0360873add.htm). Thus the sites where the overexpressed HRDC domain has been recruited remains unrepaired compared with indigenous or overexpressed full-length proteins.
Table 1 Percentage of γ-H2AX foci positive cells where cells were separately transfected with GFP-tagged full-length WRN, full-length BLM, WRN-HRDC and BLM-HRDC after MMC or MMS treatment and after required incubation, they were allowed to recover and were subjected to indirect immunolabelling for γH2AX (see text for detail)
Student's t test was used to compare γ-H2AX foci positive cells between pEGFPC1-WRN cells and pEGFPC1-WRN-HRDC cells, pEGFPC1-BLM cells and pEGFPC1-BLM-HRDC cells at the time points, 0 h and 24 h respectively (*P<0.05, **P<0.01, ***P<0.001).
Table 2 Percentage of foci positive cells
HeLa cells were separately transfected with GFP-tagged full-length WRN, full-length BLM, WRN-HRDC, BLM-HRDC and treated with either MMC or MMS. After treatment and after 24 h of recovery, numbers of foci positive cells were counted (see text for detail). Student's t test was used to compare foci positive cells between pEGFPC1-WRN cells and pEGFPC1-WRN-HRDC cells, pEGFPC1-BLM cells and pEGFPC1-BLM-HRDC cells at both the time points, 0 h and 24 h respectively (*P<0.05, **P<0.01, ***P<0.001).
3.3. Retention of damage in WHRDC and BHRDC overexpressed cells compared with the full-length WRN and BLM proteins
To address whether DNA damage induced by MMC or MMS persists for HRDC overexpressing cells, the alkaline comet assay was used under similar experimental conditions. As expected, the degree of unrepaired DNA was greater when cells were transfected with HRDC domain of WRN or BLM followed by MMC treatment (Figure 3A). Furthermore, the parameters measured, such as tail DNA, olive tail moment and tail length, are plotted in Figures 3(B)–3(D) respectively. All the parameters for cells transfected with HRDC domain were much higher after 24 h of recovery compared with their respective full-length proteins. Similar results were also observed for cells treated with MMS (Figure 4).
The exact role of WRN and BLM in DNA damage-response pathways are not yet understood. The distinct domains of these two proteins have been associated with different DNA metabolism in vitro, but their in vivo roles are not known. The two proteins actively participate in DNA dsbs repair and BER. Both WRN and BLM stimulate DNA polymerase β, enhancing base incorporation during BER (Harrigan et al., 2003). WRN (Dianova et al., 2001) and BLM (Sharma et al., 2003) also strongly stimulate FEN-1 (flap endonuclease 1), which cleaves 5′ protruding flaps generated by strand displacement DNA synthesis during LP-BER (long-patch BER). In vitro evidence also indicates that WRN exonuclease acts as a proofreading enzyme for DNA polymerase β during LP-BER (Harrigan et al., 2006, 2007). In DNA dsbs, WRN has been associated with proteins involved in both HR (homologous recombination) (Otterlei et al., 2006) and NHEJ (non-homologous end joining) (Cooper et al., 2000), whereas BLM is mainly involved in HR (Wu et al., 2001). Live cell imaging after laser-induced damage indicated that both the HRDC domain of WRN (Lan et al., 2005) and BLM (Karmakar et al., 2006) are recruited to DNA dsbs. Here, we report for the first time that WRN and BLM also respond to DNA damage, other than DNA dsbs, through their HRDC domain. Although the HRDC domain of WRN has a putative nucleic acid binding motif, crystal structure analysis show that the HRDC domain lacks any DNA binding motif and is very stable (Kitano et al., 2007). Our observation suggests that the HRDC domain of WRN or BLM is directly recruited to DNA damage sites and co-localization with γ-H2AX. γ-H2AX is an indicator of the DNA damage re<@?show=[fo]?>sponse. γ-H2AX is an important factor for DNA dsb repair, but there is also evidence that γ-H2AX forms foci in response to the damaging agents, e.g. MMS or MMNG (N-methyl-N′-nitro-N-nitrosoguanidine) or H2O2, that produce a significant amount of SSBs (single-strand breaks) (Zhou et al., 2006, Staszewski et al., 2008, Grudzenski et al., 2010). One step further, to verify that full-length WRN, BLM or their HRDC domains form foci other than dsbs, control experiments were performed in which PCNA was used as a marker of DNA damage and H2O2 as the damaging agent that produces significant SSBs. H2O2 (250 μM, 30 min) induced distinct PCNA foci that co-localized with the exogenous full-length WRN, BLM or their HRDC domains separately (Supplementary Figure S4 available at http://www.cellbiolint.org/cbi/036/cbi0360873add.htm). Although it may be possible that they are not directly associated with damage DNA, they can be recruited to the damage sites by other proteins. Among the two damaging agents, MMC-induced DNA cross-linking that ultimately uses dsbs repair pathway, but MMS, being an alkylating agent, can induce base damage that is repaired by the BER pathway. Thus both the HRDC domain of WRN and BLM may be actively involved in the initial processing of damaged DNA. Their recruitment is either direct contact with DNA or additional proteins for their association in the vicinity of damaged DNA are needed.
The physical and functional interactions of different proteins with both WRN and BLM indicate their essential role in DNA repair. In our case, the HRDC domain was sufficient in the initial step of DNA damage processing, but recruitment of other proteins through this domain is limited. Thus the complete repair process is absent in cells transfected with the HRDC domain of WRN and BLM. As a result these cells are sensitive to DNA damaging agents compared with the cells transfected with their full-length counterpart. Additionally, the role of full-length protein in DNA repair came directly from comet assay. Together, our observation demonstrates that the HRDC domain of both WRN and BLM is recruited to the DNA damage sites induced by MMC and MMS, but the catalytic function of the full-length proteins is necessary for successful repair.
In conclusion, the HRDC domain of WRNp and BLMp is recruited to laser microirradiation-induced DNA dsbs immediately after DNA damage. DNA damage produced by MMC or MMS is also recognized by the HRDC domain of these two proteins. But for processing of damage and/or repair events, the other domain of these two proteins is essential.
Saheli Samanta mainly did the laboratory work, prepared the Figures and legends, and helped in writing the manuscript. Parimal Karmakar originated the idea and wrote the manuscript.
We thank Dr C.K. Panda, CNCI (Chittaranranjan National Cancer Research Institute), Kolkata, India for the comet analysis software facility. We also thank Professor Yasui (Sendai, Japan) for providing the constructs.
This work was supported by the
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Received 21 September 2011/10 May 2012; accepted 1 June 2012
Published as Cell Biology International Immediate Publication 1 June 2012, doi:10.1042/CBI20110510
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Determination of cellular survival after transfection of cells with full-length WRN or BLM or their respective HRDC domain following treatment with MMC and MMS
Figure 2 Intracellular localization of WRN-HRDC and BLM-HRDC after DNA damage induced by MMC and MMS
Figure 3 Persistence of damage in WRN-HRDC and BLM-HRDC overexpressed cells compared with the full-length WRN and BLM proteins after MMC treatment