Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40

DNA Repair 3 (2004) 1175–1185

Review

Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40 Mitsuyoshi Yamazoe∗ , Eiichiro Sonoda, Helfrid Hochegger, Shunichi Takeda CRESTO, The Japan Science and Technology Corporation, Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan Available online 28 April 2004

Abstract In the ‘post-genome’ era, reverse genetics is one of the most informative and powerful means to investigate protein function. The chicken B lymphocyte line DT40 is widely used for reverse genetics because the cells have a number of advantages, including efficient gene targeting as well as a remarkably stable phenotype. Furthermore, the absence of functional p53 in DT40 cells enables identification of DNA damage using chromosome analysis by suppressing damage-induced apoptosis during interphase. This review summarizes the contribution of DT40 cells to reverse genetic studies of DNA damage response pathways in higher eukaryotic cells. © 2004 Elsevier B.V. All rights reserved. Keywords: DT40; Gene disruption; Conditional mutant; Homologous DNA recombination; Genome instability

1. Ontogeny of chicken B lymphocyte precursors The method of immunoglobulin variable (IgV) gene diversification and the ontogeny of B lymphocytes differ distinctively among species (reviewed in [1]). Humans and mice develop B lymphocytes in the bone marrow, where B precursors generate functional IgV segments through a site-specific process called V(D)J recombination. In contrast, rabbits and chicken develop B lymphocytes in a specific tissue, namely the appendix of the intestinal tract in rabbits and the bursa of Fabricius in chicken [1,2]. Fig. 1A illustrates the primary structure of the chicken Ig light chain locus, which carries a single V and J segment with 25 pseudo-V segments upstream of the V segments [3,4]. In the chicken B lymphocytes, the contribution of V(D)J recombination to IgV diversification is minimal because of the limited number of V and J segments. However, chicken B precursors can effectively diversify IgV segments by intragenic homologous DNA recombination (HR), called Ig gene conversion (Fig. 1B). When precursor cells proliferate in the bursa of Fabricius, they repeat Ig gene conversion events several times, with upstream pseudogenes serving as donors, leading to formation of a large IgV repertoire in preimmune

∗

Corresponding author.

1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.03.039

B lymphocytes. Rabbits, pigs and cows also diversify the IgV segment mainly through gene conversion [5,6]. Maintenance of the peripheral B lymphocyte pool also differs between avian and mammalian species. While mature B lymphocytes are continuously supplied from the bone marrow throughout life in the human and mouse, chicken peripheral B lymphocytes are maintained by self-renewal without new supply from primary lymphoid organs. The mechanism of affinity maturation of Ig is also markedly different in birds. In chicken, the Ig gene conversion is responsible not only for the formation of the primary B lymphocyte repertoire, but also the diversification of IgV segment in secondary lymphoid tissues [7]. During affinity maturation, peripheral B lymphocytes undergo Ig gene conversion in an early phase following antigenic stimulation, and subsequently diversify the IgV segments mainly through single base substitutions [7]. In contrast, human and mouse B lymphocytes employ only accumulation of base substitutions upon antigenic stimulation during affinity maturation.

2. The DT40 cell line The chicken B lymphocyte line, DT40, transformed with an avian leukosis virus [8], continuously undergoes Ig gene conversion in culture [9]. Remarkably, targeted integration

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Fig. 1. Chicken immunoglobulin light chain locus. (A) Complete organization of the chicken ␭ light-chain locus. The locus comprises a single J-C unit, a single functional V gene (V␭ 1) with a leader sequence (L) and an adjacent pool of 25 pseudogenes (␸V␭ cluster). (B) Mechanism of diversifying the variable segment of the Ig genes according to development of B cells. VJ rearrangement occurs between a single V and J segment. Afterwards the pool of pseudogenes act as donors in a process by which sequence information is mobilized by gene conversion into the single active V gene. This process can take place repeatedly.

occurs at essentially all loci in these cells with efficiencies that are orders of magnitude higher than those observed in mammalian cells [10]. This efficient gene targeting appears to reflect an intrinsic character of chicken B lymphocytes, which is shared by most analyzed chicken B lymphocyte lines and not by any non-B cell lines. The molecular mechanism responsible for the high targeting efficiencies in chicken B lymphocyte lines is not clear. Presumably, a mechanism that is involved in the Ig gene conversion also enhances gene targeting efficiencies, since both processes are mediated by HR and observed only in chicken B lymphocyte lines including DT40 cells. Gene targeting constructs contain a selection marker gene flanked by genomic sequences at both sides. The length of these sequences greatly affects the efficiency of targeting. In fact, efficient gene targeting in mouse embryonic stem (ES) cells requires more than 10 kb of the genomic sequences [11]. In contrast, DT40 cells exhibit considerably more efficient gene targeting (up to 80% of the stably transfected clones) even when shorter genomic sequences in targeting constructs are used: minimal arm lengths of ∼1.5 kb for shorter and ∼3 kb for longer arms usually result in successful targeting. It should be noted that targeting efficiency appears to depend on the length of genomic fragments that are deleted by gene targeting. For example, 110 kb and 57 kb deletions occur in only one out of 56 and 48 stable transfectants, respectively [12]. While the generation of targeting constructs has been the most time-consuming

step in the gene targeting procedure, the availability of the chicken genome database in 2004 [13], in addition to the chicken EST databases (http://www.chick.umist.ac.uk, http://genetics.hpi.uni-hamburg.de/dt40.html) [14,15], will greatly simplify gene targeting in DT40 cells and facilitate the identification of chicken ortholog genes (reviewed in [16]). Besides efficient gene targeting, DT40 cells possess a number of features that make them suitable for reverse genetic studies (reviewed in [17]). First, DT40 cells exhibit a relatively invariant character in both karyotype and phenotype even during extended periods of cell culture. This stable character is a great advantage over murine ES cells, whose multipotency is often lost during in vitro passage and after exposure to genotoxic stress. Thus, the targeting of multiple genes employing different selection markers (neo, his, bsr, puro, his, ecogpt, and zeocin) allows analysis of genetic interactions on a cellular level. Second, phenotypic analysis using cell culture assays like colony formation is greatly facilitated by the rapid growth rate of DT40 cells, which have a doubling time of 8–10 h. Third, since the cloning efficiency of wild-type cells is nearly 100%, isolation of stably transfected cells as well as subcloning of cells is easily done. Fourth, the absence of functional p53, which induces apoptosis and cell cycle arrest in the G2 phase upon DNA damage, appears to greatly contribute to successful isolation of gene-disrupted clones that exhibit genome instability, although it makes an important distinction between the DT40 and mammalian systems, which may limit extrapolation from DT40 to mammalian cells. For example, DT40 cells deficient in Atm, the protein kinase that activates the cellular response to double-strand breaks (DSBs) in the DNA, can grow at nearly normal growth kinetics even in the presence of genome instability [18], although it strongly inhibits the growth of fibroblasts derived from Atm-deficient mice [19]. Furthermore, the compromised apoptosis pathway allows us to evaluate the extent of genome instability by measuring spontaneously arising chromosomal breaks in mitotic cells, since cells can enter the M phase without stimulating the apoptosis pathway during interphase [20]. Thus, the absence of functional p53 offers a great advantage for comprehensive reverse genetic study of genome instability. Taken together, these advantages account for the wide use of DT40 cells as a cellular model for reverse genetic studies, as summarized in Table 1.

3. Methods of conditional inactivation of genes Four methods have been used successfully in DT40 cells to render cells conditionally null for essential genes (Table 2). Using these methods, genes of interest can be inactivated by specifically controlling expression or function of a targeted gene. To this end, the LoxP signal sequences or cDNA encoding a hormone-binding domain of the estrogen receptor (ER) is knocked-in into the gene of interest

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Table 1 The use of DT40 cells as a cellular model for reverse genetic studies Pathway, function, protain

Disrupted genes

DNA recombination, DNA repair, DNA replication

Rad51 [20], Rad52 [50], Rad54 [72], Rad51B [43], Rad51C, Rad51D, XRCC2, XRCC3 [44], Mre11 [53], Brca1, FANC-D2 [73], FANC-G [74], Ligase IV [75], Ku70 [52], DNA-dependent protein kinase, Rag2 [76], AID [77,78], Fen1 [79], Rad18 [28], Pol␬ [48], Rev1 [70], Rev3 [29], Xpa [48] WER, BLM [49], RecQL1, RecQL5 [80] Ubc9 [81] Atm [18,59], Nbs1 [58], Chk1 [82], Arg [83] Cyclin D1 [84], Cdh1 [85] Cleavage stimulation factor (CstF) [22], ASF/SF2 [21], TATA binding protein, cTAF(II)31, [23] MEKK1 kinase [86], CAD/DFF40 nuclease [87], Caspase 6 [88], Caspase 7 [89], Annexin 5 [90] Histone H1, H2A, H2B, h2B-V, H3-IV/H3-V, H4, Histone deacetylase-1, 2, 3 [91], histone acetyltransferase-1 (reviewed in [92]), HMG-17 [93]

RecQ DNA helicase Protein modification Damage checkpoint Cell cycle Transcription and RNA metabolism Apoptosis Histones, histone-modifying enzymes, chromatin maintenance Chromosome maintenance Heat shock factors Signal transduction

Metabolism, scavenger proteins Unknown function

CENP-C [32], CENP-H [94], CENP-I [95], Scc1/Rad21 (cohesin) [24], SMC2 (condensin) [25] Hsf1 [35], Hsf3 [34] Fyn, Syk [96], Csk [97], Btk [98], CD45 phophotase [99], PLC-␥2 [100], Abl [101], Cbl-b [102], Inositol 1, 4, 5-triphosphate receptor (type 1,2,3) [103], Blnk [104], Bam32 [105], Src homology 2 domain-containing protein-tyrosine phosphatases (SHP-1 and SHP-2) [106,107], BCAP [108] HPRT [109], thioredoxin [110] The survival of motor neurons (SMN), neutral sphingomyelinase 1 [111]

(Fig. 2A). Alternatively, a conditionally repressible gene is introduced through random integration (Fig. 2B) and the transgene is expected to keep the null cell alive by expressing the gene product (a ‘rescue construct’). A rescue construct has been made using three methods: the tetracycline (tet) inducible system, the Cre-mediated deletion system, and temperature sensitive mutant transgenes. 3.1. Tetracycline inducible promoter The tet system, which provides regulated expression of transgenes in response to varying concentrations of tetracycline in the media (Table 2), is used most frequently because of its relative tractability. Indeed, the first conditionally gene-disrupted DT40 clones were generated using the tet-Off gene expression systems by Manley and co-workers [21] who made conditionally mutant cells of a splicing fac-

tor, ASF/SF2; a polyadenylation factor, CstF [22]; and a general transcription factor, TAFII 31 [23]. We generated conditionally gene-disrupted cells with the tet system using the following protocol: first, we designed a rescue plasmid that conditionally expresses a cDNA of the gene of disruption, and gene-targeting constructs. The rescue plasmid contains the tet promoter and a transgene, followed by an internal ribosome entry site (IRES) of encephalomyocarditis virus and the luciferase gene (Fig. 2C), which enable monitoring the level of the transgene expression by measuring luciferase activity [24]. The tet system has a few technical problems. Leaky expression is detectable in a majority of the clones transfected with a rescue plasmid even after inhibition of the tet promoter. This problem can be solved by using the luciferase gene to screen clones that exhibit tight regulation of expression from the rescue plasmid. Employing a tet-dependent

Table 2 The advantages and disadvantages of the four methods of conditional inactivation of genes in the DT40 cell system Inducible system

Advantage

Disadvantage

tet-on

Suppression in a synchronous manner. Tet-dependent suppressor factor is available to inhibit leaky expression. Suppression of gene expression can be done in a synchronous manner. Endogenous promoter can be used.

Slower suppression than Tet-off.

tet-off Chimera with tamoxifen receptor Cre-recombinase Temperature sensitive (Ts) mutants

Null mutant cells can be generated. Endogenous promoter can be used. A given protein can be inactivated immediately after temperature shift. Inactivation of a given protein can be done in a reversible manner.

Slow suppression of a given protein. Leaky expression. Addition of the tamoxifen receptor may interfere with function of a protein. Available only for nuclear proteins. Deletion of genes occurs at different time points in each cell. Very difficult to generate. Characterization of each Ts mutant is difficult.

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Still another critical drawback of the tet system is the slow reduction in the amount of an analyzed protein. If mutant cells die before complete depletion of the analyzed protein, a null phenotype of the protein cannot be addressed. Furthermore, this slow inactivation might lead to an artificial phenotype in conditionally inactivated mutants. This was illustrated when we made conditionally DT40 cells deficient in Rad51, which is the eukaryotic homologue of the Escherichia coli RecA recombinase, and essential for proliferation of vertebrate cells. Inhibition of the tet promoter of a Rad51 transgene in RAD51−/− cells (hereafter descibed as rad51 cells) resulted in an inability to repair double-strand breaks that arose during DNA replication, resulting in cell cycle arrest and death within 24 h [20]. It is believed that DSBs can be caused by replication block in a sister chromatid, and are then repaired by HR, which facilitates DNA synthesis from the damaged sister using the other intact sister chromatid as a template. Thus, the absence of HR-mediated repair should leave sister chromatid breaks unrepaired in mitotic chromosomes. However, Rad51-deficient cells do not exhibit chromatid type breaks in mitotic cells, but do exhibit chromosome type breaks, where both sisters are broken at the same sites. Presumably, a reduced amount of Rad51 still initiates but does not complete HR-mediated repair, resulting in DSBs of both sisters when the chromosomes are condensed prior to the M phase. Conceivably, if Rad51 can be completely depleted before the cells undergo DNA replication, the mutant will exhibit chromatid type breaks in the subsequent M phase. This idea can be tested by a protocol developed by us whereby depletion of Rad51 and synchronization at the G1/S boundary are done in parallel [24]. 3.2. The Cre site-specific recombinase Fig. 2. Strategies for producing cells conditionally null for essential genes. After heterozygous (+/−) mutant cells are generated, two strategies (A and B) can be taken to make homozygous (−/−) mutant cells. (A) Knock-in sequences of conditional inactivation into the remaining+allele, and (B) random integration of a transgene that expresses the deleted gene product. (C) There are two types of conditionally suppressible transgenes: In the upper expression vector, the tet-mediated gene expression can be monitored by measuring luciferase activity, while in the lower vector, cre/loxP-mediated gene depletion can be monitored by flow-cytometric analysis of green fluorescent protein (GFP).

transcriptional suppressor in addition to the tet-dependent activator might further reduce the level of leaky expression. Another problem is that the tet promoter is too strong and results in gene expression far higher than the physiological level. For example, when cells are rendered deficient in a structural component of the chromosome, even a slight change in its expression in the cells might significantly reduce their viability. Indeed, Hudson et al. [25] had to screen many clones to obtain the cells that exhibit an optimal level of expression of ScII, a molecule necessary for condensation of mitotic chromosomes, from the tet promoter of a rescue plasmid.

Using an inducible Cre site-specific recombinase, MerCreMer [26], a transgene can be deleted conditionally (Fig. 2). MerCreMer carries two mutated hormone-binding domains of the estrogen receptor [26] that bind the antagonist 4-hydroxytamoxifen (TAM). This Cre-mediated recombination works efficiently so that a gene flanked by the loxP signals at both sides can be deleted in virtually all the cells within 24 h after the addition of TAM [27]. The Cre/loxP system has the following strengths and weaknesses when compared with the tet-inducible system. Cre-mediated deletion of a gene enables study of the effect of a null mutation on the cells, while the tet system cannot fully exclude the effect of leaky expression. We therefore employed the Cre/loxP system when we investigated the synthetic lethality of rad52/xrcc3 [27], rad54/rad18 [28], and rad54/rev3 cells [29]. The Cre/loxP system has the disadvantage, however, of being difficult to use for phenotypic analysis because Cre-mediated deletion does not occur in a synchronous manner in a population of cells; the tet system does work synchronously. Furthermore, both TAM and Cre site-specific recombinase have genotoxic effects to some extents [30,31] and appropriate control samples need to be

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prepared for their evaluation [28]. Another drawback of the Cre/loxP system is the absence of cre-mediated deletion in a small fraction of the cells even after their continuous exposure to TAM [27]. Despite these weaknesses, the Cre/loxP system has proved useful, especially when mutant cells exhibit genome instability and may acquire additional suppressor mutations during in vitro passage; with Cre/loxP their phenotype can be analyzed immediately after conditional inactivation of the genes. 3.3. A chimeric protein with mER and temperature sensitive mutant The ER domain is also used for the third method of generating conditional mutant cells. When Fukagawa et al. [32] disrupted a structural components of centromere, CENP-C, they found a rescue transgene could not be used because even a slight change in the amount of the CENP-C molecule in the cells significantly reduced their viability. They needed to modify the endogeneous CENP-C gene so it could be expressed from its endogeneous promoter while the encoded protein was inactivated conditionally. To this end, the entire coding region of the CENP-C gene was replaced by a chimeric cDNA encoding chicken CENP-C fused to ER. Upon the elimination of TAM, the nuclear localization of the chimeric CENP-C was gradually lost over several divisions, leading to cell death. Since the elimination of TAM does not immediately inactivate the chimeric molecule by sequstering it in the cytoplasm, the authors made another conditional mutant: DT40 cells expressing a temperature sensitive (ts) mutated CENP-C protein [33]. While the above three conditionally inactivation methods—tet, Cre/loxP system, and fusion with the ER domain—do not permit rapid inactivation of a given protein, the fourth method, ts mutation, allows the disruption of the protein function instantly by shifting the temperature of the cell culture. The physiological body temperature of birds is

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higher than that of human or mouse, ranging from 40.9 to 41.9 ◦ C, making chicken cell lines suitable for generating ts mutants. The culture temperature of DT40 cells can be varied from 34 ◦ C to as high as 43 ◦ C without loss of viability. Moreover, the response of DT40 cells to heat stress has been carefully studied by Nakai and Ishikawa [34,35]. Using this advantage, Fukagawa et al. [33] generated ts mutants of CENP-C; these cells grow normally in culture at 34 ◦ C, and shifting the temperature to 43 ◦ C caused all the cells to stop cycling in the subsequent G1 phase. Both phenotypic analysis and generation of ts mutants have a number of technical difficulties. Identification of appropriate ts mutations is extremely difficult even when the three-dimensional structure of the protein is available. Moreover, upon inactivation at a restrictive temperature, the function of many yeast ts mutated proteins is not restored after return to permissive temperatures. In addition, both the mechanism and the extent of temperature sensitive protein inactivation are distinctly different in each ts mutant and rarely understood in molecular detail. Despite these drawbacks, once an appropriate ts mutant is identified, the precise role of the protein at each phase of the cell cycle can be identified by transiently exposing a synchronized population of the cells to a restrictive temperature. The ts system, therefore, offers an enormous advantage for phenotypic analysis if a given molecule has distinct roles at different cell cycle phases. 3.4. Phenotypic analysis of essential DNA repair gene There is a caveat in the phenotypic analysis of essential genes of DNA repair pathways: inactivation of essential genes will lead to accumulation of DNA damage, stimulation of damage checkpoint, and eventually apoptosis. Thus, the direct cause of cell death must be identified while excluding any consequences of activated apoptotic processes. An inhibitor of caspases is helpful for excluding the effect

Fig. 3. Level of spontaneously arising chromosomal breaks. More than 100 mitotic cells were counted in each genotype.

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of activation of apoptosis pathways [36]. More importantly, the morphology of mitotic chromosomes is very useful for identifying the cause of cell death in most of the DNA repair mutants. DT40 cells carrying a few chromosomal breaks appear to reach metaphase without fully activating the G2 checkpoint [20]. Moreover, it is believed that a single chromosomal break in mitotic chromosomes reflects a single unrepaired DSB, which eventually leads to cell death [37]. This is also the case for DT40 cells, where there is a strong correlation between the level of spontaneously arising chromosomal breaks and the viability of each HR mutant DT40 clone (Fig. 3). Thus, analysis of chromosomal breaks is an informative way to identify the essential function of DNA repair proteins. Genome instability of yeast DNA repair mutants is difficult to evaluate because their genome is not large enough for morphological analysis of chromosomes. In contrast, in metazoan cells, chromosome analysis of gene disrupted clones provides a highly sensitive method for investigating mechanisms of maintaining chromosomal integrity, because even a single chromosomal aberration in the whole genome is detectable. Consequently, we believe that a vertebrate cellular model system for genetic analysis of genome instability is of great importance for our understanding of genome maintenance.

4. Phenotypic analysis of DNA repair mutants 4.1. Homologous DNA recombination In the budding yeast, the capability of HR can be precisely evaluated by the following assays: meiotic HR, IR sensitivity, and intragenic as well as heteroallelic HR following DSBs induced by the HO restriction enzyme [38,39]. This HO-induced HR assay allows us to analyze not only the generation of final recombination products, but also recombination intermediate molecules with time [40]. In contrast, no phenotypic assay can detect such intermediate molecules in higher eukaryotic cells. The following assays are used to measure the capability of HR in DT40 cells: (1) intragenic HR induced by I-Sce-I restriction enzyme digestion in an artificial construct [41,42]; (2) intragenic HR in the immunoglobulin light chain gene (Ig gene conversion) [9]; (3) Rad51 focus formation after IR [43,44]; (4) gene targeting efficiency [10]; (5) sister chromatid exchange (SCE) [45]; (6) sensitivity to killing by IR and crosslinking agents; and (7) spontaneously arising chromosomal breaks [46]. The I-Sce-I induced assay is also widely used in mammalian cells because it can specifically address the capability of HR and distinguish intragenic HR from HR between sister chromatids [47]. Likewise, analysis of Ig gene conversion is highly informative for specifically assessing HR, because this phenotypic assay includes determination of base sequences of HR products in the Ig V segments, permitting measurement of the length

of gene conversion tracts and the fidelity of HR reactions. In contrast, data from assays (3) through (6) above may be affected by other factors in addition to the HR capability of the mutant cells. For example, SCE reflects DNA repair mediated by HR, so the level of SCE should be determined not only by the efficiency of HR repair but also by the level of DNA damage, i.e. substrate of HR repair. Indeed, the level of SCE is increased in mutants of the translesion DNA synthesis (TLS) pathway, presumably because defective TLS may lead to an increase in single-stranded gaps, which stimulate HR-mediated repair [28,48]. Gene targeting efficiency may also be affected by the “recombinogenic character” of the chromosome, as exemplified in cells deficient in Bloom helicase (blm) cells, which show an elevation of both gene targeting efficiency and the level of SCE [49]. Nevertheless, bearing the “recombinogenic character” in mind, analysis of gene targeting efficiency is a reliable and sensitive method to analyse HR, and can be used to detect a subtle recombination defect. A reduction in gene targeting is the only detectable HR defect in rad52 cells, for example, which otherwise appear to be perfectly proficient in recombination [50]. Sensitivity to killing by genotoxic agents is determined by many factors, including various DNA repair and damage checkpoint pathways. For example, both TLS mutants, such as rad18 DT40 cells [28], and HR mutants, such as rodent xrcc3 cells [51], are sensitive to a variety of genotoxic agents, including alkylating agents, ultraviolet rays (UV), crosslinking agents including cisplatin, and IR. Thus, it is difficult to conclude which repair pathway is impaired by analyzing colony survival after genotoxic treatment. Nevertheless, IR sensitivity of synchronized populations of cells is informative to determine an impaired repair pathway. Both mammalian and chicken cells are more tolerant to IR at late S—than during early S phase. We demonstrated that this tolerance is caused by DSB repair mediated by HR between sister chromatids at late S phase, evidenced by a flat pattern of IR sensitivity during S phase seen in HR deficient cells such as rad54 or xrcc2 cells [52]. Therefore, if a gene-disrupted clone shows elevated IR sensitivity at late S phase but not at early S phase, a defect in HR-mediated DSB repair is the likely explanation. The contribution of a gene to HR can be evaluated also by IR irradiation in the G2 phase, followed by measuring chromosomal breaks in the subsequent M phase [29]. If HR capability is severely compromised, the cells exhibit extensive chromosomal breaks and are no longer able to progress through the cell cycle. Such HR mutants include rad50 (unpublished), rad51 [20], mre11 [53], nbs1 (unpublished), brca2 null cells (unpublished), and cells deficient in both Rad52 and XRCC3 [27]. Among these mutants, rad51 cells display the most severe phenotype and are not able to perform even a single round of the cell cycle. This observation indicates that Rad51 is a key factor in HR in vertebrate cells, which is in marked contrast with the budding yeast, where Rad51, Rad52, and Rad54 contribute to similar

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extents to HR. If HR capability is moderately compromised, the mutant cells will be able to proliferate while the gene deletion might still prove detrimental to embryonic development. This type of the HR mutant includes rad51 paralog cells. Fractions of these mutant cells exhibit spontaneous chromosomal breaks and cell death during the cell cycle [43,44], while xrcc2, xrcc3, and rad51d mutant mice are embryonic lethal [54]. If HR capability is only marginally compromised, the gene-disrupted mice are viable and even fertile. This type of the HR mutant includes rad52 and rad54, both of which show both reduced gene targeting efficiency while only rad54 cells are IR sensitive [55,56]. 4.2. Nonhomologous end joining There is at least one more DSB repair pathway besides HR, termed nonhomologous end-joining (NHEJ). This pathway depends on the Ku proteins, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), XRCC4, and ligase IV. While HR plays a dominant role in repairing DSBs that arise during replication, NHEJ is preferentially used for DSB repair following IR in G1 phase (reviewed in [17]). When IR induces DSBs in late S to G2 phase, NHEJ and HR have a redundant role in repairing the DSBs [52]. Interestingly, the relative contribution of the two DSB repair pathways to these cell cycle phases differs between DT40 and mammalian cells: HR is preferentially used in DT40 cells, while HR and NHEJ may contribute equally to DSB repair in mammalian cells [42]. Three phenotypic assays specifically address the capability of NHEJ in DT40 cells. (1) NHEJ-deficient cells may exhibit extremely high IR sensitivity in a population synchronized at the G1 phase but not in the G2 phase [52]. (2) An assay using linearized plasmid DNA, which contains a short direct repeat at the ends: recircularization of transfected plasmid is mediated either by using short stretches of the (micro)homology, or by using no homology at all [57]. The latter type of recircularization requires Ku70 and DNA-PKcs but not Nbs1 or Mre11 [58]. (3) Disruption of the Ku70 gene in gene-disrupted cells would be useful for investigating whether the gene acts on the same pathway as NHEJ. For example, disruption of the Ku70 gene further increased the level of IR-induced chromosomal breaks in mre11 DT40 cells, indicating the presence of a Ku70-dependent DSB repair pathway that does not depend on Mre11 [53]. Likewise, defects of both Ku70 and Atm caused a synergistic increase in IR sensitivity, indicating that Ku70 and Atm act independently of each other to prevent IR-irradiated cells from apoptosis [59]. An assay of V(D)J recombination should be developed to take advantage of the informative potential of base sequence analysis of V(D)J joints [60]. 4.3. Translesion DNA synthesis Replication blocks are released by post-replicational repair, which is carried out by two major pathways, HR and

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TLS (reviewed in [61,62]). There are three classic phenotypic assays that evaluate TLS in the budding yeast. First, budding yeast is transformed with duplex plasmids carrying adducts, such as an N-2-acetylaminofluorene adduct within a GGG or GGCG sequence, in one strand, followed by analysis of the replication of each strand [63,64]. The second assay is determination of induced mutation frequencies following exposure of cells to a diverse array of mutagens. In addition, the level of spontaneously arising base substitutions may be affected by TLS pathways, because the majority of spontaneous mutations are generated by Pol␨ and Rev1p in the budding yeast (reviewed in [65]). The third assay measures the size of newly replicated DNA before and after UV-irradiation by velocity sedimentation analyses in alkaline sucrose gradients [66,67]. This assay can detect defective TLS post-UV damage in human cells derived from a variant form of xeroderma pigmentosum (XP-V) (reviewed in [68]). We have not yet established the first and second assays for DT40 cells, or obtained reliable positive data from rad18 and rev3 cells using the third assay. The most prominent phenotype of TLS-deficient DT40 cells is extremely high sensitivity to crosslinking agents. Indeed, rev3 cells show higher sensitivity to cisplatin than any other DNA repair mutants, including cells deficient in Fanconi genes (unpublished data), HR, and the nucleotide excision repair pathway [29]. TLS mutants derived from DT40 and murine ES cells, such as rad18, rev1, polκ, and polζ, also exhibit elevated sensitivity to a variety of genotoxic agents, including H2 O2 , N-2-acetylaminofluorene, benzo[␣]pyrene, alkylating agents, crosslinking agents, IR, and UV [29,69,70]. Furthermore, defective TLS increases UV-induced chromosomal breaks, particularly when cells are exposed to UV at early S-phase [28]. Another prominent phenotype of TLS-deficient DT40 mutants is increased levels of spontaneous as well as induced SCE, which may reflect upregulation of HR-mediated post-replicational repair to compensate for defective TLS [28,29,48]. Although a phenotypic assay that can specifically address the capability of TLS has not yet been established in DT40 cells, combined analysis of SCE, UV-induced chromosomal breaks, and sensitivity to a variety of genotoxic agents may be sufficient to confirm a defect in TLS. Of note, there is a caveat in the phenotypic analysis of TLS mutants: since the Rev3 DNA polymerase and Rad18 are involved in both TLS and HR, the interpretation of the phenotype of these mutants is complicated by this dual function [28,29].

5. Forward genetics 5.1. Screening of suppressor genes of ts mutants of CENP-C Fukagawa et al. [33] successfully identified suppressor genes of the CENP-C ts mutant. To efficiently introduce a large number of cDNAs into a population of DT40 cells, they generated a cDNA library using a retrovirus expression

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vector. Purified DNA of the cDNA library was transfected into about 108 of the virus packaging MP34 cell line [71], which can produce recombinant retrovirus. Following transient expression of the recombinant retrovirus library in the MP34 cells, several liters of virus-containing supernatant were subjected to ultracentrifugation twice to enrich virus particles by about 1000-fold. They eventually yielded media with surprisingly high titers of virus, 1010−12 pfu/ml, which corresponds to about one virus hit in every DT40 cell. For infection, 107 DT40 cells were exposed to 1 ml of the concentrated virus supernatants. Fukagawa et al. screened cDNAs that can rescue the CENP-C ts mutants at the restrictive temperature, and successfully isolated several suppressor genes (personal communication). Interestingly, these genes have previously not been implicated in any function associated with the centromere. This study demonstrates that DT40 mutants are extremely useful for screening large numbers of cDNAs in a suppressor screen. Since many nonviable mutants have already been generated from DT40 cells, this new protocol will be used to identify genes that can rescue these mutants.

6. Conclusion Yeast species have great advantages over vertebrate cells for genetic studies. Indeed, forward genetic studies of yeast not only identified genes involved in HR but also contributed to the identification of their mammalian orthologs. Although these studies have revealed a molecular mechanism of HR, accumulating evidence indicates that the contribution of each ortholog protein to the HR reaction is not necessarily the same in yeast and mammalian cells. For example, rad51, rad52, and rad54 mutants exhibit similar phenotypes in the budding yeast. In marked contrast, although depletion of Rad51 is lethal to the mammalian cells, mice deficient in Rad52 or Rad54 are viable and have no developmental defect. To compare the relative role of each ortholog protein, DT40 cells are a unique system among higher eukaryotic cells for comprehensive reverse genetic analysis of gene function. Defects in DNA repair pathways including HR cause genome instability leading to stimulation of damage checkpoints. Thus, genome instability often dramatically reduces the viability of the murine fetus as well as its primary cultured cells. Even if mutant mice are viable, their primary culture cells are not very useful materials on which to investigate chromosomal DNA transactions because of their limited growth capability. Thus, the analysis of a given transformed cell line is informative for analyzing fundamental cellular function that is shared by virtually every lineage of cells, such as DNA repair pathways. Extensive comparisons of phenotypes of HR-deficient mutants have revealed that there is no significant discrepancy between murine ES and DT40 cells. DT40 cells therefore have been widely employed for re-

verse genetic studies, despite obvious concerns about the use of a transformed cell line, which may have certain cell line-specific phenotypic characteristics. Analysis of DT40 cells will also contribute to the development of methods to increase gene targeting in human cell culture systems.

Acknowledgements Financial support was provided in part by CREST. JST. (Saitama, Japan) and the Center of Excellence (COE) grant for Scientific Research from the Ministry of Education, Culture, Sports and Technology and by grants from The Uehara Memorial Foundation, and The Naito Foundation. This work was funded in part by grants from the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.

References [1] C.A. Reynaud, B. Bertocci, A. Dahan, J.C. Weill, Formation of the chicken B-cell repertoire: ontogenesis, regulation of Ig gene rearrangement, and diversification by gene conversion, Adv. Immunol. 57 (1994) 353–378. [2] R.S. Becker, K.L. Knight, Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits, Cell 63 (1990) 987–997. [3] C.A. Reynaud, V. Anquez, A. Dahan, J.C. Weill, A single rearrangement event generates most of the chicken immunoglobulin light chain diversity, Cell 40 (1985) 283–291. [4] C.A. Reynaud, V. Anquez, H. Grimal, J.C. Weill, A hyperconversion mechanism generates the chicken light chain preimmune repertoire, Cell 48 (1987) 379–388. [5] J.E. Butler, J. Sun, I. Kacskovics, W.R. Brown, P. Navarro, The VH and CH immunoglobulin genes of swine: implications for repertoire development, Vet. Immunol. Immunopathol. 54 (1996) 7–17. [6] M.R. Lucier, R.E. Thompson, J. Waire, A.W. Lin, B.A. Osborne, R.A. Goldsby, Multiple sites of V lambda diversification in cattle, J. Immunol. 161 (1998) 5438–5444. [7] H. Arakawa, K. Kuma, M. Yasuda, S. Furusawa, S. Ekino, H. Yamagishi, Oligoclonal development of B cells bearing discrete Ig chains in chicken single germinal centers, J. Immunol. 160 (1998) 4232–4241. [8] T.W. Baba, B.P. Giroir, E.H. Humphries, Cell lines derived from avian lymphomas exhibit two distinct phenotypes, Virology 144 (1985) 139–151. [9] J.M. Buerstedde, C.A. Reynaud, E.H. Humphries, W. Olson, D.L. Ewert, J.C. Weill, Light chain gene conversion continues at high rate in an ALV-induced cell line, EMBO J. 9 (1990) 921–927. [10] J.M. Buerstedde, S. Takeda, Increased ratio of targeted to random integration after transfection of chicken B cell lines, Cell 67 (1991) 179–188. [11] C. Deng, M.R. Capecchi, Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus, Mol. Cell Biol. 12 (1992) 3365–3371. [12] Y. Takami, S. Takeda, T. Nakayama, An approximately half set of histone genes is enough for cell proliferation and a lack of several histone variants causes protein pattern changes in the DT40 chicken B cell line, J. Mol. Biol. 265 (1997) 394–408. [13] D. Burt, O. Pourquie, Genetics chicken genome-science nuggets to come soon, Science 300 (2003) 1669.

M. Yamazoe et al. / DNA Repair 3 (2004) 1175–1185 [14] I. Abdrakhmanov, D. Lodygin, P. Geroth, H. Arakawa, A. Law, J. Plachy, B. Korn, J.M. Buerstedde, A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function, Genome Res. 10 (2000) 2062–2069. [15] P.E. Boardman, J. Sanz-Ezquerro, I.M. Overton, D.W. Burt, E. Bosch, W.T. Fong, C. Tickle, W.R. Brown, S.A. Wilson, S.J. Hubbard, A comprehensive collection of chicken cDNAs, Curr. Biol. 12 (2002) 1965–1969. [16] D.F. Hudson, C. Morrison, S. Ruchaud, W.C. Earnshaw, Reverse genetics of essential genes in tissue-culture cells: dead cells talking, Trends Cell Biol. 12 (2002) 281–287. [17] E. Sonoda, C. Morrison, Y.M. Yamashita, M. Takata, S. Takeda, Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (2001) 111–117. [18] N. Takao, H. Kato, R. Mori, C. Morrison, E. Sonada, X. Sun, H. Shimizu, K. Yoshioka, S. Takeda, K. Yamamoto, Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation, Oncogene 18 (1999) 7002–7009. [19] Y. Xu, D. Baltimore, Dual roles of ATM in the cellular response to radiation and in cell growth control, Genes Dev. 10 (1996) 2401– 2410. [20] E. Sonoda, M.S. Sasaki, J.-M. Buerstedde, O. Bezzubova, A. Shinohara, H. Ogawa, M. Takata, Y. Yamaguchi-Iwai, S. Takeda, Rad51 deficient vertebrate cells accumulate chromosomal breaks prior to cell death, EMBO J. 17 (1998) 598–608. [21] R. Lemaire, J. Prasad, T. Kashima, J. Gustafson, J.L. Manley, R. Lafyatis, Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2: a novel function for SR proteins, Genes Dev. 16 (2002) 594–607. [22] Y. Takagaki, J.L. Manley, Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation, Mol. Cell 2 (1998) 761–771. [23] Z. Chen, J.L. Manley, Robust mRNA transcription in chicken DT40 cells depleted of TAF(II)31 suggests both functional degeneracy and evolutionary divergence, Mol. Cell Biol. 20 (2000) 5064–5076. [24] E. Sonoda, T. Matsusaka, C. Morrison, P. Vagnarelli, O. Hoshi, T. Ushiki, K. Nojima, T. Fukagawa, I.C. Waizenegger, J.M. Peters, W.C. Earnshaw, S. Takeda, Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells, Dev. Cell 1 (2001) 759–770. [25] D.F. Hudson, P. Vagnarelli, R. Gassmann, W.C. Earnshaw, Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes, Dev. Cell 5 (2003) 323–336. [26] Y. Zhang, J. Wienands, C. Zurn, M. Reth, Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP-65 and Syk, EMBO J. 17 (1998) 7304–7310. [27] A. Fujimori, S. Tachiiri, E. Sonoda, L.H. Thompson, P.K. Dhar, M. Hiraoka, S. Takeda, Y. Zhang, M. Reth, M. Takata, Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells, EMBO J. 20 (2001) 5513–5520. [28] Y.M. Yamashita, T. Okada, T. Matsusaka, E. Sonoda, G.Y. Zhao, K. Araki, S. Tateishi, M. Yamaizumi, S. Takeda, RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells, EMBO J. 21 (2002) 5558–5566. [29] E. Sonoda, T. Okada, G.Y. Zhao, S. Tateishi, K. Araki, M. Yamaizumi, T. Yagi, N.S. Verkaik, D.C. van Gent, M. Takata, S. Takeda, Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates, EMBO J. 22 (2003) 3188–3197. [30] A. Loonstra, M. Vooijs, H.B. Beverloo, B.A. Allak, E. van Drunen, R. Kanaar, A. Berns, J. Jonkers, Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 9209–9214.

1183

[31] D.P. Silver, D.M. Livingston, Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity, Mol. Cell 8 (2001) 233–243. [32] T. Fukagawa, C. Pendon, J. Morris, W. Brown, CENP-C is necessary but not sufficient to induce formation of a functional centromere, EMBO J. 18 (1999) 4196–4209. [33] T. Fukagawa, V. Regnier, T. Ikemura, Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells, Nucleic Acids Res. 29 (2001) 3796–3803. [34] A. Nakai, T. Ishikawa, Cell cycle transition under stress conditions controlled by vertebrate heat shock factors, EMBO J. 20 (2001) 2885–2895. [35] A. Nakai, T. Ishikawa, A nuclear localization signal is essential for stress-induced dimer-to-trimer transition of heat shock transcription factor 3, J. Biol. Chem. 275 (2000) 34665–34671. [36] E.J. Brown, D. Baltimore, ATR disruption leads to chromosomal fragmentation and early embryonic lethality, Genes Dev. 14 (2000) 397–402. [37] L.C. Huang, K.C. Clarkin, G.M. Wahl, Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 4827–4832. [38] F. Paques, J.E. Haber, Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae, Microbiol. Mol. Biol. Rev. 63 (1999) 349–404. [39] J.E. Haber, Uses and abuses of HO endonuclease, Methods Enzymol. 350 (2002) 141–164. [40] N. Sugawara, X. Wang, J.E. Haber, In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination, Mol. Cell 12 (2003) 209–219. [41] R.D. Johnson, N. Liu, M. Jasin, Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination, Nature 401 (1999) 397–399. [42] T. Fukushima, M. Takata, C. Morrison, R. Araki, A. Fujimori, M. Abe, K. Tatsumi, M. Jasin, P.K. Dhar, E. Sonoda, T. Chiba, S. Takeda, Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair, J. Biol. Chem. 276 (2001) 44413–44418. [43] M. Takata, M.S. Sasaki, E. Sonoda, T. Fukushima, C. Morrison, J.S. Albala, S.M. Swagemakers, R. Kanaar, L.H. Thompson, S. Takeda, The Rad51 paralog Rad51B promotes homologous recombinational repair, Mol. Cell Biol. 20 (2000) 6476–6482. [44] M. Takata, M.S. Sasaki, S. Tachiiri, T. Fukushima, E. Sonoda, D. Schild, L.H. Thompson, S. Takeda, Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs, Mol. Cell Biol. 21 (2001) 2858–2866. [45] E. Sonoda, M.S. Sasaki, C. Morrison, Y. Yamaguchi-Iwai, M. Takata, S. Takeda, Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells, Mol. Cell Biol. 19 (1999) 5166–5169. [46] E. Sonoda, M. Takata, Y.M. Yamashita, C. Morrison, S. Takeda, Homologous DNA recombination in vertebrate cells, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 8388–8394. [47] R.D. Johnson, M. Jasin, Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells, EMBO J. 19 (2000) 3398–3407. [48] T. Okada, E. Sonoda, Y.M. Yamashita, S. Koyoshi, S. Tateishi, M. Yamaizumi, M. Takata, O. Ogawa, S. Takeda, Involvement of vertebrate Polkappa in Rad18-independent post-replication repair of UV damage, J. Biol. Chem. 27 (2002) 27. [49] W. Wang, M. Seki, Y. Narita, E. Sonoda, S. Takeda, K. Yamada, T. Masuko, T. Katada, T. Enomoto, Possible association of BLM in decreasing DNA double strand breaks during DNA replication, EMBO J. 19 (2000) 3428–3435. [50] Y. Yamaguchi-Iwai, E. Sonoda, J.-M. Buerstedde, O. Bezzubova, C. Morrison, M. Takata, A. Shinohara, S. Takeda, Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52, Mol. Cell Biol. 18 (1998) 6430–6435.

1184

M. Yamazoe et al. / DNA Repair 3 (2004) 1175–1185

[51] N. Liu, J.E. Lamerdin, R.S. Tebbs, D. Schild, J.D. Tucker, M.R. Shen, K.W. Brookman, M.J. Siciliano, C.A. Walter, W. Fan, L.S. Narayama, Z.-Q. Zhou, A.W. Adamson, K.J. Sorensen, D.J. Chen, N.J. Jones, L.H. Thompson, XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA crosslinks and other damages, Mol. Cell 1 (1998) 783–793. [52] M. Takata, M.S. Sasaki, E. Sonoda, C. Morrison, M. Hashimoto, H. Utsumi, Y. Yamaguchi-Iwai, A. Shinohara, S. Takeda, Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells, EMBO J. 17 (1998) 5497–5508. [53] Y. Yamaguchi-Iwai, E. Sonoda, M.S. Sasaki, C. Morrison, T. Haraguchi, Y. Hiraoka, Y.M. Yamashita, T. Yagi, M. Takata, C. Price, N. Kakazu, S. Takeda, Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells, EMBO J. 18 (1999) 6619–6629. [54] B. Deans, C.S. Griffin, P. O’Regan, M. Jasin, J. Thacker, Homologous recombination deficiency leads to profound genetic instability in cells derived from xrcc2-knockout mice, Cancer Res. 63 (2003) 8181–8187. [55] T. Rijkers, J. Van Den Ouweland, B. Morolli, A.G. Rolink, W.M. Baarends, P.P. Van Sloun, P.H. Lohman, A. Pastink, Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation, Mol. Cell Biol. 18 (1998) 6423–6429. [56] J. Essers, H. van Steeg, J. de Wit, S.M. Swagemakers, M. Vermeij, J.H. Hoeijmakers, R. Kanaar, Homologous and non-homologous recombination differentially affect DNA damage repair in mice, EMBO J. 19 (2000) 1703–1710. [57] N.S. Verkaik, R.E. Esveldt-van Lange, D. van Heemst, H.T. Bruggenwirth, J.H. Hoeijmakers, M.Z. Zdzienicka, D.C. van Gent, Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells, Eur. J. Immunol. 32 (2002) 701–709. [58] H. Tauchi, J. Kobayashi, K. Morishima, D.C. Van Gent, T. Shiraishi, N.S. Verkaik, D. VanHeems, E. Ito, A. Nakamura, E. Sonoda, M. Takata, S. Takeda, S. Matsuura, K. Komatsu, Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells, Nature 420 (2002) 93–98. [59] C. Morrison, E. Sonoda, N. Takao, A. Shinohara, K. Yamamoto, S. Takeda, The controlling role of ATM in homologous recombinational repair of DNA damage, EMBO J. 19 (2000) 463–471. [60] D. Moshous, I. Callebaut, R. de Chasseval, B. Corneo, M. Cavazzana-Calvo, F. Le Deist, I. Tezcan, O. Sanal, Y. Bertrand, N. Philippe, A. Fischer, J.P. de Villartay, Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency, Cell 105 (2001) 177–186. [61] J.E. Haber, DNA repair. Gatekeepers of recombination, Nature 398 (1999) 665–667. [62] H. Flores Rozas, R.D. Kolodner, Links between replication, recombination and genome instability in eukaryotes, Trends Biochem. Sci. 25 (2000) 196–200. [63] K. Baynton, A. Bresson-Roy, R.P. Fuchs, Analysis of damage tolerance pathways in Saccharomyces cerevisiae: a requirement for Rev3 DNA polymerase in translesion synthesis, Mol. Cell Biol. 18 (1998) 960–966. [64] K. Baynton, A. Bresson-Roy, R.P. Fuchs, Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae, Mol. Microbiol. 34 (1999) 124–133. [65] C.W. Lawrence, Cellular roles of DNA polymerase zeta and Rev1 protein, DNA Repair (Amst.) 1 (2002) 425–435. [66] L. Prakash, Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations, Mol. Gen. Genet. 184 (1981) 471–478.

[67] S. Tateishi, Y. Sakuraba, S. Masuyama, H. Inoue, M. Yamaizumi, Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 7927–7932. [68] A.R. Lehmann, Replication of damaged DNA in mammalian cells: new solutions to an old problem, Mutat. Res. 509 (2002) 23–34. [69] T. Ogi, Y. Shinkai, K. Tanaka, H. Ohmori, Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 15548– 15553. [70] L.J. Simpson, J.E. Sale, Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line, EMBO J. 22 (2003) 1654–1664. [71] T. Yoshimatsu, M. Tamura, S. Kuriyama, K. Ikenaka, Improvement of retroviral packaging cell lines by introducing the polyomavirus early region, Hum. Gene Ther. 9 (1998) 161–172. [72] O. Bezzubova, A. Silbergleit, Y. Yamaguchi-Iwai, S. Takeda, J.M. Buerstedde, Reduced X-ray resistance and homologous recombination frequencies in a RAD54−/− mutant of the chicken DT40 cell line, Cell 89 (1997) 185–193. [73] C.J. Vandenberg, F. Gergely, C.Y. Ong, P. Pace, D.L. Mallery, K. Hiom, K.J. Patel, BRCA1-independent ubiquitination of FANCD2, Mol. Cell 12 (2003) 247–254. [74] K. Yamamoto, M. Ishiai, N. Matsushita, H. Arakawa, J.E. Lamerdin, J.M. Buerstedde, M. Tanimoto, M. Harada, L.H. Thompson, M. Takata, Fanconi anemia FANCG protein in mitigating radiationand enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells, Mol. Cell Biol. 23 (2003) 5421– 5430. [75] N. Adachi, T. Ishino, Y. Ishii, S. Takeda, H. Koyama, DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: implications for DNA double-strand break repair, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 12109–12113. [76] S. Takeda, E.L. Masteller, C.B. Thompson, J.M. Buerstedde, RAG-2 expression is not essential for chicken immunoglobulin gene conversion, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 4023–4027. [77] R.S. Harris, J.E. Sale, S.K. Petersen-Mahrt, M.S. Neuberger, AID is essential for immunoglobulin V gene conversion in a cultured B cell line, Curr. Biol. 12 (2002) 435–438. [78] H. Arakawa, J. Hauschild, J.M. Buerstedde, Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion, Science 295 (2002) 1301–1306. [79] Y. Matsuzaki, N. Adachi, H. Koyama, Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H(2)O(2), Nucleic Acids Res. 30 (2002) 3273–3277. [80] W. Wang, M. Seki, Y. Narita, T. Nakagawa, A. Yoshimura, M. Otsuki, Y. Kawabe, S. Tada, H. Yagi, Y. Ishii, T. Enomoto, Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation, Mol. Cell Biol. 23 (2003) 3527–3535. [81] T. Hayashi, M. Seki, D. Maeda, W. Wang, Y. Kawabe, T. Seki, H. Saitoh, T. Fukagawa, H. Yagi, T. Enomoto, Ubc9 is essential for viability of higher eukaryotic cells, Exp. Cell Res. 280 (2002) 212–221. [82] G. Zachos, M.D. Rainey, D.A. Gillespie, Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects, EMBO J. 22 (2003) 713–723. [83] Y. Li, H. Shimizu, S.L. Xiang, Y. Maru, N. Takao, K. Yamamoto, Arg tyrosine kinase is involved in homologous recombinational DNA repair, Biochem. Biophys. Res. Commun. 299 (2002) 697– 702. [84] J.M. Lahti, H. Li, V.J. Kidd, Elimination of cyclin D1 in vertebrate cells leads to an altered cell cycle phenotype, which is rescued by overexpression of murine cyclins D1, D2, or D3 but not by a mutant cyclin D1, J. Biol. Chem. 272 (1997) 10859–10869. [85] T. Sudo, Y. Ota, S. Kotani, M. Nakao, Y. Takami, S. Takeda, H. Saya, Activation of Cdh1-dependent APC is required for G1 cell

M. Yamazoe et al. / DNA Repair 3 (2004) 1175–1185

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93] [94]

[95]

[96]

[97]

[98]

[99]

cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells, EMBO J. 20 (2001) 6499–6508. R. Kwan, J. Burnside, T. Kurosaki, G. Cheng, MEKK1 is essential for DT40 cell apoptosis in response to microtubule disruption, Mol. Cell Biol. 21 (2001) 7183–7190. K. Samejima, S. Tone, W.C. Earnshaw, CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis, J. Biol. Chem. 276 (2001) 45427–45432. S. Ruchaud, N. Korfali, P. Villa, T.J. Kottke, C. Dingwall, S.H. Kaufmann, W.C. Earnshaw, Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation, EMBO J. 21 (2002) 1967–1977. N. Korfali, S. Ruchaud, D. Loegering, D. Bernard, C. Dingwall, S.H. Kaufmann, W.C. Earnshaw, Caspase-7 gene disruption reveals an involvement of the enzyme during the early stages of apoptosis, J. Biol. Chem. 28 (2003) 28. T.E. Hawkins, D. Das, B. Young, S.E. Moss, DT40 cells lacking the Ca2+ -binding protein annexin 5 are resistant to Ca2+ -dependent apoptosis, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 8054–8059. Y. Takami, R. Nishi, T. Nakayama, Histone H1 variants play individual roles in transcription regulation in the DT40 chicken B cell line, Biochem. Biophys. Res. Commun. 268 (2000) 501–508. T. Nakayama, Y. Takami, Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure, J. Biochem. (Tokyo) 129 (2001) 491–499. Y. Li, J.B. Dodgson, The chicken HMG-17 gene is dispensable for cell growth in vitro, Mol. Cell Biol. 15 (1995) 5516–5523. T. Fukagawa, Y. Mikami, A. Nishihashi, V. Regnier, T. Haraguchi, Y. Hiraoka, N. Sugata, K. Todokoro, W. Brown, T. Ikemura, CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells, EMBO J. 20 (2001) 4603– 4617. A. Nishihashi, T. Haraguchi, Y. Hiraoka, T. Ikemura, V. Regnier, H. Dodson, W.C. Earnshaw, T. Fukagawa, CENP-I is essential for centromere function in vertebrate cells, Dev. Cell 2 (2002) 463–476. M. Takata, H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, T. Kurosaki, Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways, EMBO J. 13 (1994) 1341–1349. A. Hata, H. Sabe, T. Kurosaki, M. Takata, H. Hanafusa, Functional analysis of Csk in signal transduction through the B-cell antigen receptor, Mol. Cell Biol. 14 (1994) 7306–7313. M. Takata, T. Kurosaki, A role for Bruton’s tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2, J. Exp. Med. 184 (1996) 31–40. S. Yanagi, H. Sugawara, M. Kurosaki, H. Sabe, H. Yamamura, T. Kurosaki, CD45 modulates phosphorylation of both autophosphorylation and negative regulatory tyrosines of Lyn in B cells, J. Biol. Chem. 271 (1996) 30487–30492.

1185

[100] M. Takata, Y. Homma, T. Kurosaki, Requirement of phospholipase C-gamma 2 activation in surface immunoglobulin M-induced B cell apoptosis, J. Exp. Med. 182 (1995) 907–914. [101] N. Takao, R. Mori, H. Kato, A. Shinohara, K. Yamamoto, c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance, J. Biol. Chem. 275 (2000) 725–728. [102] T. Yasuda, T. Tezuka, A. Maeda, T. Inazu, Y. Yamanashi, H. Gu, T. Kurosaki, T. Yamamoto, Cbl-b positively regulates Btk-mediated activation of phospholipase C-gamma2 in B cells, J. Exp. Med. 196 (2002) 51–63. [103] H. Sugawara, M. Kurosaki, M. Takata, T. Kurosaki, Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor, EMBO J. 16 (1997) 3078–3088. [104] C. Fu, C.W. Turck, T. Kurosaki, A.C. Chan, BLNK: a central linker protein in B cell activation, Immunity 9 (1998) 93–103. [105] H. Niiro, A. Maeda, T. Kurosaki, E.A. Clark, The B lymphocyte adaptor molecule of 32 kD (Bam32) regulates B cell antigen receptor signaling and cell survival, J. Exp. Med. 195 (2002) 143– 149. [106] A. Maeda, M. Kurosaki, M. Ono, T. Takai, T. Kurosaki, Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal, J. Exp. Med. 187 (1998) 1355–1360. [107] H. Okada, S. Bolland, A. Hashimoto, M. Kurosaki, Y. Kabuyama, M. Iino, J.V. Ravetch, T. Kurosaki, Role of the inositol phosphatase SHIP in B cell receptor-induced Ca2+ oscillatory response, J. Immunol. 161 (1998) 5129–5132. [108] T. Okada, A. Maeda, A. Iwamatsu, K. Gotoh, T. Kurosaki, BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation, Immunity 13 (2000) 817–827. [109] T. Fukagawa, N. Hayward, J. Yang, C. Azzalin, D. Griffin, A.F. Stewart, W. Brown, The chicken HPRT gene: a counter selectable marker for the DT40 cell line, Nucleic Acids Res. 27 (1999) 1966– 1969. [110] T. Tanaka, F. Hosoi, Y. Yamaguchi-Iwai, H. Nakamura, H. Masutani, S. Ueda, A. Nishiyama, S. Takeda, H. Wada, G. Spyrou, J. Yodoi, Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis, EMBO J. 21 (2002) 1695–1703. [111] A.C. Fensome, M. Josephs, M. Katan, F. Rodrigues-Lima, Biochemical identification of a neutral sphingomyelinase 1 (NSM1)-like enzyme as the major NSM activity in the DT40 B-cell line: absence of a role in the apoptotic response to endoplasmic reticulum stress, Biochem. J. 365 (2002) 69–77.