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in tolerance of endogenous and exogenous DNA damage in mouse B cells
1 Laboratory for Antigen Receptor Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan
2 Division of Pathology, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan
3 Evolutionary Genetics, HMRO Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
4 Department of Developmental Genetics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
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Abstract |
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(Pol) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Pol in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Pol polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, 
-irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Pol is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Pol in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes. |
Introduction |
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(Pol) is a family A polymerase with a unique structure (Harris et al. 1996; Sharief et al. 1999) and is the only known DNA polymerase that contains an intrinsic helicase domain. Pol was first identified in Drosophila as the mus308 gene product (Harris et al. 1996). Mus308-deficient mutant flies exhibited hypersensitivity to DNA interstrand cross-linking agents such as cisplatin, but not to the alkylating agent methyl methanesulfonate (MMS) (Boyd et al. 1990). These observations suggested a specific role for mus308 product in repair of interstrand cross-links (ICLs) in Drosophila. However, a role for mus308 product in repair of other types of DNA lesions has not been explored.
Human and mouse Pol have an overall structure similar to the fly Pol, with a helicase domain in the N-terminal region and a polymerase domain in the C-terminus (Seki et al. 2003; Shima et al. 2003). Biochemical analysis using purified recombinant human Pol (POLQ) revealed that POLQ has extremely low fidelity, catalyzing considerable misincorporation when copying non-damaged DNA (Seki et al. 2004). Moreover, POLQ was able to bypass an abasic site and a thymine glycol but unable to bypass either cisplatin-induced 1,2-d(GpG) and 1,3-d(GpTpG) adducts or UV-induced cyclobutane pyrimidine dimmer (CPD) and 6-4 photoproduct (6-4 PP). A unique and unusual property of POLQ is its ability to efficiently catalyze both the insertion and extension steps for bypass of an abasic site (Seki et al. 2004). Consistent with the presence of conserved helicase motifs, POLQ exhibited a single-stranded DNA-dependent ATPase activity (Seki et al. 2003).
The function of Pol in mammalian cells remains poorly understood. In a phenotype-based mutagenesis screen for chromosome instability mutants in mice, Shima et al. (2003) identified a recessive mutation called chaos1 (chromosome aberration occurring spontaneously), which exhibited elevated levels of spontaneous and radiation-induced micronuclei in erythrocytes. The chaos1 mutation was found to map to the Polq gene, which contained a T-to-C base substitution causing a serine-to-proline change in amino acid residue 1932. These investigators recently generated Pol-deficient mice, which reportedly exhibit a very similar phenotype to chaos1, thus confirming the identity of Polq and chaos1 (Shima et al. 2004). Surprisingly, apart from increased micronuclei in erythrocytes, Pol-deficient mice and embryo fibroblasts (MEFs) did not show any obvious abnormalities and displayed the same sensitivity as WT mice and MEFs to -irradiation and the DNA crosslinking agent mitomycin C (MMC).
Pol orthologs have been identified in mammals including mouse and human but not in lower eukaryotes such as yeast or in prokaryotes. This suggests that Pol has evolved relatively recently and may have a more specialized function. Consistent with this possibility, we found that human and mouse Polq exhibit a tissue-specific expression pattern, with preferential expression in lymphoid tissues and testis, where it may play a role in DNA metabolism during spermatogenesis (Kawamura et al. 2004). Most interestingly, abundant Polq transcripts were detected in germinal center (GC) B cells where somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes occur (Kawamura et al. 2004).
The lymphoid tissue- and stage-specific expression pattern of Polq in both human and mouse suggested an important role for Pol in mammalian lymphocytes. In the present study, we have inactivated Pol polymerase activity in CH12 mouse B lymphoma cells, which, like GC B cells, express a high level of Pol and undergo Ig gene CSR upon stimulation (Kunimoto et al. 1992; Nakamura et al. 1996). Analysis of mutant CH12 cells devoid of the polymerase activity revealed that mammalian Pol plays an important role in DNA repair not only of ICLs but also a variety of other DNA lesions. Moreover, we show that Pol is involved in tolerance of MMS through a mechanism that appears to be different from base excision repair (BER).
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Results |
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polymerase activity
To inactivate the DNA polymerase activity of Pol, we decided to delete exon 25, which contains a highly conserved aspartic acid (Asp) residue known to be essential for the catalytic activity of family A polymerases (Patel & Loeb 2000; Marini et al. 2003). Substitution of this Asp with alanine has been shown to completely abrogate the polymerase activity of both E. coli PolI and human POLN. Since CH12 cells have three copies of chromosome 16 where Polq is localized, we designed two types of targeting vectors to sequentially delete exon 25 on the three alleles (Fig. 1A,B). The targeting vector 1 was constructed to replace exons 25 and 26 with a neo gene and the targeting vector 2 was designed to delete only exon 25 after Cre-mediated excision (Fig. 1C).
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25,26), while in the remaining allele, only exon 25 was deleted (25).
The deletion of both exons 25 and 26 (185-bp and 112-bp, respectively) or exon 25 alone should result in the production of an 8.3–8.4 kb mutant mRNA, which is nearly identical to the size of WT Polq mRNA (8.6 kb). Northern blot analysis indeed revealed a similar size mRNA in WT, KO and TKOflox cells (data not shown). To further verify the correct targeting of the three alleles, we performed RT-PCR analysis using primers flanking exons 25 and 26 (primers s6623 and as7597, see Fig. 1B), and the expected bands were observed in mutant cells at each stage of targeting (Fig. 2A). We further gel-purified and sequenced each of these bands, and confirmed that the 
1 kb band was derived from WT mRNA, the 800-bp band lacked exon 25 and the 700-bp band lacked both exons 25 and 26 (Fig. 2A). A faint 1.1 kb band observed in KOneo and DKOneo was also sequenced and found to be derived from an aberrantly spliced mRNA (exon 24 spliced to a pseudo-splice acceptor sequence within the intron between exon 24 and exon 25), suggesting that replacement of exons 25 and 26 with a neo gene affected the splicing between exon 24 and exon 27. The absence of WT mRNA in TKOflox suggests that the targeted allele 2 (flox, Fig. 1C) could not be correctly spliced, possibly due to the presence of the neo gene directly upstream of exon 25.
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protein encoded by exons 1 through 24 (plus 11 amino acids encoded by the incorrect reading frame of exon 26). The deletion of both exons 25 and 26 (25,26) should lead to an inframe message that theoretically could produce an mRNA composed of exon 1 through 24 plus exon 27 through 30. This would encode a mutant protein with a similar MW as WT Pol. Indeed, immunoprecipitation and Western blot analysis revealed an immunoreactive protein in the mutant cells with a molecular weight similar to the WT Pol (Fig. 2B). We could not detect a truncated protein encoded by exons 1–24 because the antibodies recognize a peptide sequence located on exon 30 near the C-terminal region. These results indicate that TKO cells express a mutant protein lacking exons 25 and 26.
CH12-TKO have greatly reduced Pol polymerase activity
To verify that TKO are indeed devoid of polymerase activity, we next analyzed template-dependent DNA polymerase activity of immunopurified WT and mutant Pol (Fig. 2B). As shown in Fig. 2C, WT Pol (lane 5) exhibited DNA polymerase activity. In contrast, mutant Pol (lane 7) had no detectable polymerase activity. The Pol polymerase activity was much lower than that observed with 1 unit of either Klenow (Fig. 2C, lane 1) or exonuclease free Klenow (Fig. 2C, lane 2). In this regard, it is interesting to note that Drosophila Pol, when immunoprecipitated with antiserum against the central linker domain, exhibited far greater (approximately 50-fold) DNA polymerase activity than when immunoprecipitated with antiserum against the polymerase domain (Pang et al. 2005). These investigators concluded that the binding of the antibodies to the polymerase domain inhibited (or neutralized) Pol catalytic activity. Because the antibodies we used in immunoprecipitation recognize a peptide sequence located at the C-terminus of the polymerase domain, the relatively weak activity we observed could be due to the inhibitory effect caused by the antibody binding to the polymerase domain. We conclude that TKO are largely devoid of Pol polymerase activity.
Pol-deficient cells exhibit a slightly reduced growth rate and increased spontaneous cell death
We first examined the growth properties of WT and TKO. To avoid any subtle differences in culture conditions that might affect cell growth, we carefully seeded the cells at the same density in fresh medium each time of cell passage. In addition, before we seeded the cells for proliferation analysis, we eliminated dead cells by the density separation medium Lympholyte M. We found that TKO had a slightly reduced growth rate compared to WT cells (Fig. 3A,B). Although the reduction was rather modest, it was highly reproducible and statistically significant (P = 0.017 and P = 0.035, Student's t-test). Figure 3A shows the results of six independent experiments with WT and TKO (clone 15). To exclude the possibility that this difference simply reflects clonal variation, we randomly picked five TKO clones and compared their growth rate with two different stocks of WT cells that had been maintained for different periods in culture. Again, the average growth rate of the five TKO clones was significantly reduced compared to that of the two WT cells (Fig. 3B).
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Elevated sensitivity to DNA damaging agents in Pol-deficient cells
Pol has been implicated in repair of DNA ICLs in Drosophila as the fly mus308 mutants were highly sensitive to DNA crosslinking agents such as cisplatin (Boyd et al. 1990). We therefore examined the sensitivity of WT, KO, DKO and two TKO clones (cl.7 and cl.15) to MMC and cisplatin, both of which generate ICLs. Consistent with the observations in Drosophila, TKO were more sensitive to these agents than were WT cells (Fig. 4A,B). In addition, TKO exhibited increased sensitivity to etoposide (Fig. 4C) and -irradiation (Fig. 4D), which induce DNA double-strand breaks (DSBs). UV sensitivity was also increased in TKO (Fig. 4E). Unexpectedly, the Pol-deficient cells demonstrated elevated sensitivity to the alkylating agent MMS (Fig. 4F), to which Drosophila mus308 mutants did not show increased sensitivity (Boyd et al. 1990). Although there was some variation, KO and DKO usually exhibited sensitivity to these treatments that was intermediate between WT and TKO (Fig. 4). These results demonstrate that the polymerase activity of Pol is involved in tolerance of not only DNA ICLs and DSBs but also UV adducts and alkylation damage in mammalian cells.
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DNA damage induced by alkylating agents such as MMS is thought to be repaired primarily by the BER pathway in mammalian cells. The elevated MMS sensitivity of TKO therefore suggested a potential role for Pol in BER. Mammalian BER consists of two subpathways, the predominant "single-nucleotide or short patch BER" and the alternate "long patch BER" (Dianov et al. 1992; Klungland & Lindahl 1997; Wilson 1998). The single-nucleotide BER strictly depends on the 5'-deoxyribosephosphate (5'dRP) lyase activity of DNA polymerase ß (Polß) (Sobol et al. 2000) and is strongly inhibited by methoxyamine (MX), which binds to the abasic site formed by glycosylase-mediated excision of the damaged base and blocks Polß-mediated removal of 5'dRP (Horton et al. 2000). To explore a possible involvement of Pol in BER, we examined MMS sensitivity of WT and TKO in the presence of MX. Compared to WT cells, TKO exhibited higher MMS sensitivity in the absence of MX, as revealed by the representation of either the SubG1 fraction (Fig. 5A) or dead cells (Fig. 5B). Treatment with MX increased MMS sensitivity in both WT and TKO in a dose-dependent manner (Fig. 5A,B), suggesting that inhibition of the major BER pathway rendered both cell types more susceptible to MMS. However, the difference in MMS sensitivity between WT and TKO remained almost constant at each dose of MX examined. Therefore, the increased MMS sensitivity in TKO is unlikely due to a defect in the major BER pathway.
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Discussion |
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polymerase domain reported here, we have recently successfully generated Pol-null mutant CH12 cells, demonstrating that the sequential dilution method we employed is highly efficient and reproducible.
We used CH12 mouse B lymphoma cells for disruption of the Polq gene because these cells, like GC B cells, express abundant Polq mRNA and Pol protein. Analyses of the mutant cells devoid of Pol polymerase activity revealed that Pol is involved in the general maintenance of genome integrity, a finding consistent with earlier observations that Pol-deficient MEFs exhibited elevated chromosomal abnormalities (Shima et al. 2004). However, in contrast to the previous finding that Pol-deficient and WT MEFs showed the same sensitivity to -irradiation and MMC treatment (Shima et al. 2004), TKO CH12 cells were more sensitive than WT cells to these treatments. Since Pol is mainly expressed in lymphoid tissues and the testis (Kawamura et al. 2004), the lack of increased sensitivity to these DNA damaging agents in mutant MEFs is likely due to the low level of Pol expression in these cells. Likewise, Pol-deficient mice were shown to exhibit a similar sensitivity as WT mice to -irradiation and MMC in terms of their survival after the treatments (Shima et al. 2004). This may not be surprising since the elevated sensitivity of lymphoid cells and the testis to these treatments may not directly affect the survival of Pol-deficient mice.
Pol appears to be dispensable for most cell types, but the results of the present study suggest that it plays an important role in tolerance of endogenous and exogenous DNA damage in CH12 B lymphoma cells. Pol thus functions in a cell type-specific manner, which may be partly explained by its preferential expression in lymphoid tissues. Why, then, do B cells specifically require Pol for their survival and DNA repair? One possibility is that CH12, as well as GC B cells, undergo rapid cell division and may thus demand a more efficient repair machinery than other cell types. Another possibility is that GC B cells undergo dynamic genetic alterations including SHM and CSR, which may require additional repair activities. Indeed, we have recently found that Pol plays an important role in SHM of Ig genes (Masuda et al. 2005).
TKO exhibited a decreased growth rate compared to WT cells. Our results suggest that the observed decrease in cell growth in TKO is unlikely due to a defect in cell cycle progression, but rather to an increase in apoptosis. In a separate study, we have generated Pol-deficient mice. Consistently, Pol-deficient B lymphocytes purified from mouse spleen also displayed a moderate increase in spontaneous cell death as compared to WT B cells (unpublished observation). The exact cause of the elevated spontaneous death in TKO remains to be investigated. Attempts to demonstrate an increase in chromosomal aberrations in TKO, including chromatid and chromosome breaks, have been unsuccessful. It is conceivable that the absence of Pol polymerase activity may affect the repair of physiological DNA damage, leading to the accumulation of unrepaired DNA lesions and/or increased chromosome damage, which might trigger cell death.
TKO exhibited increased sensitivity to DNA interstrand crosslinking agents compared to WT cells. While cisplatin sensitivity of mus308 mutants was more than tenfold higher than WT flies (Boyd et al. 1990), we only observed an approximately twofold increase in the apoptotic cells in TKO. This discrepancy could be due to differences in the experimental conditions. In Drosophila, the larvae were fed with cisplatin-containing water and the number of surviving adults was used as an indicator for cisplatin sensitivity. In this case, any chromosomal damage induced by cisplatin, even in the absence of cell death, could potentially abort the larvae development. Therefore, the cisplatin sensitivity observed in Drosophila reflects all types of cisplatin-induced genotoxic effects. It is also possible that repair of ICLs is more dependent on the helicase and other unknown functions of Pol and the mutant Pol devoid of the polymerase activity may still retain substantial function in repair of ICLs. In this regard, it will be of interest to analyze the cisplatin and MMC sensitivity in Pol-null CH12 cells.
We have previously shown that human POLQ expression is up-regulated in lung, gastric and colon cancers as compared with matched non-tumorous tissue counterparts (Kawamura et al. 2004). Interestingly, up-regulation of POLQ expression in these cancers was associated with a poor clinical outcome (Kawamura et al. 2004). The present study revealed that Pol indeed plays a role in tolerance of DNA crosslinking agents in mammalian cells. These observations collectively suggest an intriguing possibility that tumor cells over-expressing POLQ may be more resistant to chemotherapy with DNA crosslinking agents such as cisplatin and MMC, leading to a poor clinical prognosis. We have generated several tumor lines ectopically expressing Pol and will examine their tumorigenecity in vivo in the presence of various anti-cancer agents including interstrand crosslinking agents.
Pol-deficient cells showed elevated sensitivity to -irradiation and etoposide, both of which induce DSBs. DSBs are known to be repaired by two pathways, the error-free homologous recombination (HR) and the error-prone non-homologous end joining (NHEJ) (Ferguson & Alt 2001; Valerie & Povirk 2003; Wyman et al. 2004). In mammalian cells, NHEJ is considered to be the major pathway for repair of DSBs (Valerie & Povirk 2003). Our results, together with previous observations that chaos1 mice displayed elevated levels of chromosome damage (Shima et al. 2004), collectively suggest a role for Pol in DSB repair in mammalian cells.
TKO also showed an increased sensitivity to UV irradiation compared to WT cells. Major UV adducts include CPD and 6-4 PP and are known to be repaired by NER. CPD could also be efficiently bypassed by Pol
-mediated translesion DNA synthesis (Masutani et al. 2000). Since recombinant POLQ was unable to catalyze DNA synthesis across either CPD or 6-4 PP (Seki et al. 2004), it is unclear at this point how Pol polymerase activity might be involved in repair of UV damage. An analogous situation has been observed for Pol
, which cannot bypass UV adducts under in vitro conditions but nevertheless Pol-deficient embryo fibroblasts showed increased UV sensitivity (Ogi et al. 2002).
In contrast to the normal MMS sensitivity in mus308 mutants, TKO showed a higher MMS sensitivity than WT cells, which persisted in the presence of an inhibitor for the major BER pathway. This implicates that the MMS hypersensitivity in TKO is not caused by a defect in the major BER pathway. Pol might be involved in tolerance of MMS through HR-mediated repair since recent studies suggested that MMS-induced base damage was initially tolerated by HR (Sobol et al. 2003; McNees et al. 2005). Alternatively, Pol may tolerate MMS by directly replicating over abasic sites generated during BER since it is the only enzyme that can efficiently bypass these lesions (Seki et al. 2004). Such abasic sites would otherwise stall the ongoing replication fork and trigger cell death. Further studies are required to fully understand the mechanisms by which Pol participates in repair of alkylation damage in mammalian cells.
In summary, the present study demonstrates previously unidentified roles for Pol in preventing spontaneous apoptosis and in tolerance of a variety of DNA lesions in mouse lymphocytes. It will be interesting and informative to compare the phenotype of TKO described in the present study with our recently generated Pol-null CH12 cells. Our recent finding that Pol contributes to the generation of C/G mutations during somatic hypermutation of Ig genes (Masuda et al. 2005) suggests a broad role for Pol in mammals, both in tolerance of endogenous and exogenous DNA damage and in the highly specialized immune system.
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Experimental procedures |
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A mouse cDNA fragment encoding the Pol polymerase core domain was amplified and used as a probe to screen a 129 mouse strain genomic library (Clontech, Palo Alto, CA, USA) and isolate Polq genomic clones. Two targeting vectors were constructed to sequentially disrupt the polymerase core domain (Fig. 1). In the first targeting vector, a 3.7 kb PvuII fragment and a 2.9 kb SphI fragment were used as 5'- and 3' homology regions, respectively, to replace exons 25 and 26 with the neomycin (neo) gene. In the second targeting vector, we used a 3.3 kb PvuII fragment that mostly overlapped with the replaced region of the first targeting vector in the 3' homology to avoid re-targeting of the already targeted allele, and to delete exon 25. The neo gene in both constructs was flanked by loxP sites to allow its removal by Cre recombinase.
Establishment of CH12 cells deficient in Pol polymerase catalytic activity
CH12 cells have a relatively stable near diploid karyotype except for several chromosomal fusions and translocations. There are three copies of chromosome 16 as revealed by chromosome painting analysis (unpublished results). Since Polq is located on chromosome 16, it was necessary to disrupt the polymerase domains on three alleles of the Polq gene. We first transfected 2 x 107 CH12 cells with 30 µg of the linearized 1st targeting vector. Two days later the transfected cells were cultured in the presence of 600 µg/mL G418 and 2 µM GANC for 7–10 days, during which period the dead cells were removed by the density separation medium Lympholyte M (CEDARLANE, Ontario, Canada) every 2–3 days. Because the targeting efficiency in CH12 cells is low, we employed a sequential dilution method as described below to isolate cells in which homologous recombination had occurred correctly. The G418 and GANC resistant cells were first seeded at 300 cells per well in a flat bottom 96 well plate. After cells grew to near confluence, half of the cells were frozen and the remaining cells were lyzed to extract genomic DNA using an automated DNA extraction robot (Magnia 2000; TOYOBO, Japan). Homologous recombination was initially screened for by long PCR using 5' (s2 and neo/s) and 3' (as2 and neo/as) primer sets (Fig. 1B). Two wells that contained correctly targeted cells were then expanded and each seeded at 20 cells per well in a 96 well plate. Wells that contained correctly targeted cells were similarly identified by long PCR and seeded again at 0.6 cell/well in a 96 well flat bottom plate. At this point, wells that were found by microscopic analysis to contain a single cell were marked and genotyped after expansion. At the conclusion of the sequential dilution process, we had obtained multiple clones derived from both of the initial positive wells (IC7 and IH5). The replacement of exons 25 and 26 by the neo gene was confirmed by Southern blot analysis. These cells were referred as KOneo (for single knock out).
To replace exons 25 and 26 on the second allele with the neo gene, two KOneo clones derived from the IC7 and IH5 wells were each cultured in the presence of 6 mg/mL of G418 for 2 weeks during which period the dead cells were similarly removed by Lympholyte M. The cells that survived the high concentration of G418 were then expanded and subjected to Southern blot analysis, which revealed a 4.4 kb mutant band (targeted allele 1, Fig. 1B) that had approximately twice the intensity as that in KOneo (Fig. 1C, compare KOneo and DKOneo), suggesting that the second allele had also been replaced by the neo gene. These cells were referred as DKOneo (for double knock out). Before disrupting the third allele, DKOneo cells derived from IC7 and IH5 were each transiently transfected with a vector expressing an EGFP-Cre fusion protein to excise the neo gene. Two days after transfection, the EGFP positive cells were single cell sorted, expanded, genotyped by PCR with primer set s1 and as4 (Fig. 1B), and finally the excision of the neo gene was confirmed by Southern blot hybridization (Fig. 1C). These cells were referred to as DKO. To disrupt the third allele, DKO were transfected with targeting vector 2 and screened in a similar way to obtain cells that were correctly targeted (TKOflox, for triple knock out). Finally, the floxed neo gene and exon 25 were excised by transient expression of the EGFP-Cre and single cell sorted. We ultimately obtained clones 2, 4, 5, 6 and 7 from IC7, and clones 9, 10, 11, 12, 15 and 16 from IH5. These cells were named TKO (for triple knock out).
PCR analyses
The following primers were used in genomic or RT-PCR analyses: s1, 5'-CTTTGACAGGAGCCAGAGTT-3'; s2, 5'-GGGTTGTTTGTAGGCACTGA-3'; as2, 5'-ATCGGCAGGTAGGCATCATC-3'; as4, 5'-ACAAGCCTCTCAGCAGCACA-3'; as5, 5'-ATGCAGATTCAGGAGAGGAG-3'; neo/s, 5'-TCGCCTTCTATCGCCTTCTT-3'; neo/as, 5'-ATAGCCGAATAGCCTCTCCA-3'; s6623, 5'-AGCGGGAAAAGCACCTGAAC-3'; as7597, 5'-CGTCGTGAAGTTGAAGGATG-3'. The genomic PCR was performed under the following conditions: 95°C for 2 min followed by 30 cycles of amplification at 95 °C for 5 s, 60 °C for 10 s and 72 °C for 5–10 min (depending on the sizes of PCR products) using LA-Taq (TAKARA BIO, Japan). RNA extraction and cDNA synthesis were as previously described. RT-PCR was performed at 95 °C for 2 min followed by 30 cycles of amplification at 95 °C for 5 s, 58 °C for 10 s and 72 °C for 2 min using Taq polymerase (TOYOBO, Japan).
Immunoprecipitation and polymerase assay
Polyclonal rabbit antibodies were generated against a peptide (QNDRTGLLPKRKLKG) located near the C terminus of mouse Pol. For immunoprecipitation, 2 x 107 cells were incubated for 30 min on ice in 0.5 mL of lysis buffer as described (Seki et al. 2004). The lysate was centrifuged for 10 min at 15 000 r.p.m. and the supernatant was precleared 4 times with 0.5 mL of protein A beads. The precleared lysate was incubated with 10 µL of either anti-Pol- or control rabbit IgG-conjugated protein A beads at 4 °C for 4 h and washed 5 times with the lysis buffer. Half of the washed beads (5 µL) were used for polymerase assay and the remaining half for Western blot analysis. The polymerase assay was performed essentially as described (Seki et al. 2004).
Southern, Northern and Western blot analyses
For Southern blot analysis, genomic DNA (20 µg) was digested with HindIII, resolved in a 0.7% agarose gel and transferred to a nylon membrane. A 32P-labeled SpeI-KpnI fragment (Fig. 1B) was used as a probe. Northern blot analysis was performed using a SnaI-Xho1 Polq cDNA fragment as a probe. Western blot analysis was performed as previously described (O-Wang et al. 2001) except that the lysate was prepared according to the protocol by Seki et al. (2004).
Cell growth assay and sensitivity to DNA damaging agents
For the growth assay, cells were seeded at either 1 x 104/mL or 2 x 104/mL in 12 well plates (1 mL/well). The live and dead cells were counted 48 h later using a hemocytometer and Trypan Blue exclusion. DNA content was analyzed using propidium iodide as previously described (Bahar et al. 2002) to reveal the subG1 fraction. Apoptosis was analyzed using an Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). For sensitivity to DNA damaging agents, cells were seeded at 1 x 104/mL in the presence or absence of different doses of the test agents. Initially we counted the cells with a hemocytometer. However, for samples treated with higher doses of DNA damaging agents, the cell concentration was too low to be accurately determined by this method. We therefore instead analyzed the subG1 fraction which we found correlated very well with cell death. For -ray irradiation, a 137Cs irradiator was used. UV irradiation was performed with two 8 W UVC lamps. The UV dose at 254 nm was determined with a UVX Radiometer. For analyzing the effect of methoxyamine (MX) on MMS sensitivity, cells were seeded at 5 x 104/mL, a sufficient concentration that allowed us to analyze both the subG1 fraction and the ratio of live and dead cells with an automated cell counting system (Guava PCA System, GUAVA Technologies, Hayward, CA, USA).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: oh{at}rcai.riken.jp
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References |
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Boyd, J.B., Sakaguchi, K. & Harris, P.V. (1990) mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease. Genetics 125, 813–819.[Abstract]
Dianov, G., Price, A. & Lindahl, T. (1992) Generation of single-nucleotide repair patches following excision of uracil residues from DNA. Mol. Cell. Biol.
12, 1605–1612.
Faili, A., Aoufouchi, S., Gueranger, Q., et al. (2002) AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line. Nature Immunol. 3, 815–821.[CrossRef][Medline]
Feederle, R., Delecluse, H.J., Rouault, J.P., Schepers, A. & Hammerschmidt, W. (2004) Efficient somatic gene targeting in the lymphoid human cell line DG75. Gene 343, 91–97.[CrossRef][Medline]
Feldhaus, A.L., Klug, C.A., Arvin, K.L. & Singh, H. (1993) Targeted disruption of the Oct –2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation. EMBO J. 12, 2763–2772.[Medline]
Ferguson, D.O. & Alt, F.W. (2001) DNA double strand break repair and chromosomal translocation: lessons from animal models. Oncogene 20, 5572–5579.[CrossRef][Medline]
Friedberg, E.C., Wagner, R. & Radman, M. (2002) Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science
296, 1627–1630.
Grawunder, U., Zimmer, D., Fugmann, S., Schwarz, K. & Lieber, M.R. (1998) DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol. Cell 2, 477–484.[CrossRef][Medline]
Harris, P.V., Mazina, O.M., Leonhardt, E.A., Case, R.B., Boyd, J.B. & Burtis, K.C. (1996) Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Mol. Cell. Biol. 16, 5764–5771.[Abstract]
Horton, J.K., Prasad, R., Hou, E. & Wilson, S.H. (2000) Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair. J. Biol. Chem.
275, 2211–2218.
Hubscher, U., Maga, G. & Spadari, S. (2002) Eukaryotic DNA polymerases. Annu. Rev. Biochem. 71, 133–163.[CrossRef][Medline]
Kawamura, K., Bahar, R., Seimiya, M., et al. (2004) DNA polymerase theta is preferentially expressed in lymphoid tissues and upregulated in human cancers. Int. J. Cancer 109, 9–16.[CrossRef][Medline]
Klungland, A. & Lindahl, T. (1997) Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 16, 3341–3348.[CrossRef][Medline]
Kunimoto, D.Y., Ritzel, M. & Tsang, M. (1992) The roles of IL-4, TGF-beta and LPS in IgA switching. Eur. Cytokine Netw. 3, 407–415.[Medline]
Kunkel, T.A. (2003) Considering the cancer consequences of altered DNA polymerase function. Cancer Cell 3, 105–110.[CrossRef][Medline]
Marini, F., Kim, N., Schuffert, A. & Wood, R.D. (2003) POLN, a nuclear PolA family DNA polymerase homologous to the DNA cross-link sensitivity protein Mus308. J. Biol. Chem.
278, 32014–32019.
Masuda, K., Ouchida, R., Takeuchi, A., et al. (2005) DNA polymerase contributes to the generation of C/G mutations during somatic hypermutation of Ig genes. Proc. Natl. Acad. Sci. USA
102, 13986–13991.
Masutani, C., Kusumoto, R., Iwai, S. & Hanaoka, F. (2000) Mechanisms of accurate translesion synthesis by human DNA polymerase eta. EMBO J. 19, 3100–3109.[CrossRef][Medline]
McNees, C.J., lan, L.A., Tenis, N. & Heierhorst, J. (2005) ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage. EMBO J. 24, 2447–2457.[CrossRef][Medline]
Moreadith, R.W. & Radford, N.B. (1997) Gene targeting in embryonic stem cells: the new physiology and metabolism. J. Mol. Med. 75, 208–216.[CrossRef][Medline]
Nakamura, M., Kondo, S., Sugai, M., Nazarea, M., Imamura, S. & Honjo, T. (1996) High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int. Immunol.
8, 193–201.
O-Wang, J., Kawamura, K., Tada, Y., et al. (2001) DNA polymerase kappa, implicated in spontaneous and DNA damage-induced mutagenesis, is overexpressed in lung cancer. Cancer Res.
61, 5366–5369.
Ogi, T., Shinkai, Y., Tanaka, K. & Ohmori, H. (2002) Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene. Proc. Natl. Acad. Sci. USA
99, 15548–15553.
Pang, M., McConnell, M. & Fisher, P.A. (2005) The Drosophila mus 308 gene product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear protein found in both ovaries and embryos. DNA Repair 4, 971–982.[Medline]
Patel, P.H. & Loeb, L.A. (2000) DNA polymerase active site is highly mutable: evolutionary consequences. Proc. Natl. Acad. Sci. USA
97, 5095–5100.
Seki, M., Marini, F. & Wood, R.D. (2003) POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res.
31, 6117–6126.
Seki, M., Masutani, C., Yang, L.W., et al. (2004) High-efficiency bypass of DNA damage by human DNA polymerase Q. EMBO J. 23, 4484–4494.[CrossRef][Medline]
Sharief, F.S., Vojta, P.J., Ropp, P.A. & Copeland, W.C. (1999) Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase. Genomics 59, 90–96.[CrossRef][Medline]
Shima, N., Hartford, S.A., Duffy, T., Wilson, L.A., Schimenti, K.J. & Schimenti, J.C. (2003) Phenotype-based identification of mouse chromosome instability mutants. Genetics
163, 1031–1040.
Shima, N., Munroe, R.J. & Schimenti, J.C. (2004) The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm. Mol. Cell. Biol.
24, 10381–10389.
Sobol, R.W., Kartalou, M., Almeida, K.H., et al. (2003) Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses. J. Biol. Chem.
278, 39951–39959.
Sobol, R.W., Prasad, R., Evenski, A., et al. (2000) The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity. Nature 405, 807–810.[CrossRef][Medline]
Valerie, K. & Povirk, L.F. (2003) Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22, 5792–5812.[CrossRef][Medline]
Wilson, S.H. (1998) Mammalian base excision repair and DNA polymerase beta. Mutat. Res. 407, 203–215.[Medline]
Wyman, C., Ristic, D. & Kanaar, R. (2004) Homologous recombination-mediated double-strand break repair. DNA Repair 3, 827–833.[Medline]
Yamazoe, M., Sonoda, E., Hochegger, H. & Takeda, S. (2004) Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair 3, 1175–1185.[Medline]
Received: 28 September 2005
Accepted: 1 November 2005
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