The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination.

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.

FancJ/Brip1 helicase protects against genomic losses and gains in vertebrate cells.

Defects in the FANCJ/BRIP1 helicase gene are associated with genome instability disorders such as familial breast cancer or Fanconi anemia (FA). Although FANCJ has an in vitro activity to resolve G-quadruplex (G4) structures, and FANCJ ortholog in C. elegans prevents G4-associated deletions during replication, how FANCJ loss affects genome integrity in higher organisms remains unclear. Here, we report that FANCJ, but not other FA genes FANCD2 or FANCC, protected against large-scale genomic deletion that occurred frequently at the rearranged immunoglobulin heavy chain (IgH) locus in chicken DT40 cell line, suggesting that FancJ protects the genome independently of the FA ubiquitination pathway. In a more unbiased approach using array-comparative genomic hybridization, we identified de novo deletions as well as amplifications in fancj cells kept in culture for 2 months. A cluster of G4 sequence motifs was found near the breakpoint of one amplified region, but G4 sequence motifs were not detected at the breakpoints of two deleted regions. These results collectively suggest that, unlike in C. elegans, actions of vertebrate FANCJ to promote genome stability may not be limited to protection against the G4-mediated gene deletions.

Bloom DNA helicase facilitates homologous recombination between diverged homologous sequences.

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM(-/-) cells. In addition, BLM(-/-) cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM(-/-) cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.

Cooperative roles of vertebrate Fbh1 and Blm DNA helicases in avoidance of crossovers during recombination initiated by replication fork collapse.

Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1(-)(/)(-) DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1(-)(/)(-) cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM(-)(/)(-) cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1(-)(/)(-) cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.

Role for RAD18 in homologous recombination in DT40 cells.

RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

RAD51 up-regulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors.

The breast cancer susceptibility gene BRCA1 encodes a large protein thought to contribute to a variety of cellular processes, although the critical determinants of BRCA1-deficient tumorigenesis remain unclear. Given that BRCA1 is required for cell proliferation, suppressor mutations are believed to modify BRCA1 phenotypes and contribute to the etiology of BRCA1-deficient tumors. Here, we show that overexpression of the homologous recombinase RAD51 in a DT40 BRCA1Delta/Delta mutant rescues defects in proliferation, DNA damage survival, and homologous recombination (HR). In addition, epistasis analysis with BRCA1 and the DNA end-joining factor KU70 indicates that these factors operate independently of one another to repair double-strand breaks. Consistent with this genetic finding, cell synchronization studies show that the ability of BRCA1 to promote radioresistance is restricted to the late S and G2 phases of the cell cycle, as predicted for genes whose function is specific to homology-mediated repair rather than nonhomologous end-joining. Notably, retrospective analyses of microarray expression data reveal elevated expression of RAD51 and two of its late-acting cofactors, RAD54 and RAD51AP1, in BRCA1-deficient versus sporadic breast tumors. Taken together, our results indicate that up-regulation of HR provides a permissive genetic context for cells lacking BRCA1 function by circumventing its requirement in RAD51 subnuclear assembly. Furthermore, the data support a model in which enhanced HR activity contributes to the etiology of BRCA1-deficient tumors.

ATP-induced shrinkage of DNA with MukB protein and the MukBEF complex of Escherichia coli.

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-gammaS, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.

Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination.

BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3' end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.

Critical roles for polymerase zeta in cellular tolerance to nitric oxide-induced DNA damage.

Nitric oxide (NO), a signal transmitter involved in inflammation and regulation of smooth muscle and neurons, seems to cause mutagenesis, but its mechanisms have remained elusive. To gain an insight into NO-induced genotoxicity, we analyzed the effect of NO on a panel of chicken DT40 clones deficient in DNA repair pathways, including base and nucleotide excision repair, double-strand break repair, and translesion DNA synthesis (TLS). Our results show that cells deficient in Rev1 and Rev3, a subunit essential for DNA polymerase zeta (Polzeta), are hypersensitive to killing by two chemical NO donors, spermine NONOate and S-nitroso-N-acetyl-penicillamine. Mitotic chromosomal analysis indicates that the hypersensitivity is caused by a significant increase in the level of induced chromosomal breaks. The data reveal the critical role of TLS polymerases in cellular tolerance to NO-induced DNA damage and suggest the contribution of these error-prone polymerases to accumulation of single base substitutions.

DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation.

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein.

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N6-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA71-181), and found that SeqA71-181 specifically binds to hemimethylated DNA containing a sequence with a mismatched mA:G base pair [G-mA(:G)-T-C] as efficiently as the normal hemimethylated G-mA(:T)-T-C sequence. We determined the crystal structures of SeqA71-181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 A resolutions, respectively, and found that the mismatched mA:G base pair and the normal mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.

Extensive chromosomal breaks are induced by tamoxifen and estrogen in DNA repair-deficient cells.

Tamoxifen (TAM) possesses antiestrogen activity and is widely used for the treatment or prevention of breast cancer. However, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. To explore the molecular mechanisms of TAM-induced mutagenesis, we analyzed the effects of this drug on gene-disrupted chicken B lymphocyte (DT40) clones deficient in various DNA repair pathways. Rad18, Rev3, and Polkappa are involved in translesion DNA synthesis (TLS), which facilitates recovery from replication blocks on damaged template strands. DT40 cells deficient in TLS were found to be hypersensitive to TAM, exhibiting an increase in chromosomal breaks. Furthermore, these mutants were also hypersensitive to 4-hydroxyestradiol, a physiological metabolite of estrogen. These data suggest a contribution of TLS to the prevention of chromosomal breaks by TAM and estrogen, and they therefore indicate that such error-prone DNA synthesis underlies mutagenesis induced by these agents.

Conserved domains in the chicken homologue of BRCA2.

Germline mutation of the BRCA2 gene causes a high risk of developing breast and ovarian cancer. Although the BRCA2 protein has been implicated in homologous recombination (HR) of DNA and in transcription, it is still unclear how mutation leads to tumorigenesis. We have identified a non-mammalian homologue of BRCA2 from chicken, which encodes a protein with 3397 amino acids (aa) and shows only 40% identity to human BRCA2. However, comparison of the mammalian and chicken sequences revealed remarkably high homology in several segments. These include a N-terminal region (approximately 100 aa), which was previously shown to possess intrinsic transcriptional activity, and a C-terminal region (aa residue approximately 2480-approximately 3180 in human BRCA2), which has not been clearly assigned any function. In contrast, although the eight BRC repeats of mammalian BRCA2 are believed to play an important role in HR by interacting with Rad51, the BRC3, BRC5, and BRC6 repeats exhibit virtually no similarity to their mammalian counterparts. Among 311 missense mutations listed as unclassified variants in the NIH Breast Cancer Information Core database, only 83 of these sites are identical in chicken BRCA2. Thus, chicken BRCA2 may provide a means to identify domains and residues associated with cancer predisposition.

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

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.

Compensatory functions and interdependency of the DNA-binding domain of BRCA2 with the BRCA1-PALB2-BRCA2 complex.

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2(Δ)(N) mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2(Δ)(N) mutant cells displayed a considerably milder phenotype than did BRCA2(-/-) null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2(ΔN) that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2(Δ)(N) cells. Remarkably, although the resulting BRCA2(Δ)(DBD) cells displayed a moderate phenotype, the BRCA2(Δ)(N+ΔDBD) cells displayed a very severe phenotype, as did the BRCA2(-/-) cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene.

Cells deficient in the FANC/BRCA pathway are hypersensitive to plasma levels of formaldehyde.

Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 micromol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients.

Generation of medaka gene knockout models by target-selected mutagenesis.

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization.

The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro.

Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli.

To demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.