Hiroshi Arakawa

Hiroshi Arakawa
FIRC Institute of Molecular Oncology Foundation

About

98
Publications
12,987
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7,544
Citations
Citations since 2016
6 Research Items
1792 Citations
2016201720182019202020212022050100150200250300
2016201720182019202020212022050100150200250300
2016201720182019202020212022050100150200250300
2016201720182019202020212022050100150200250300

Publications

Publications (98)
Article
Full-text available
Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, an...
Article
Full-text available
ESCO1/2 acetyltransferases mediating SMC3 acetylation and sister chromatid cohesion (SCC) are differentially required for genome integrity and development. Here we established chicken DT40 cell lines with mutations in ESCO1/2, SMC3 acetylation, and the cohesin remover WAPL. Both ESCO1 and ESCO2 promoted SCC, while ESCO2 was additionally and specifi...
Article
While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in...
Article
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All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system—gene conversion, somatic hypermutation and class switch recombination—require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential interm...
Article
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The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously imp...
Article
In eukaryotic cells helix-distorting DNA lesions like cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs) are efficiently removed by nucleotide excision repair (NER). NER is a multistep process where in the end, subsequent to replication over the gap, the remaining nick is sealed by a DNA ligase. Lig1 has been...
Article
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Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at...
Article
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Replication fork-associated factors promote genome integrity and protect against cancer. Mutations in the DDX11 helicase and the ESCO2 acetyltransferase also cause related developmental disorders classified as cohesinopathies. Here we generated vertebrate model cell lines of these disorders and cohesinopathies-related genes. We found that vertebrat...
Article
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Author Summary During the B cell immune response, immunoglobulin (Ig) genes are subject to a unique mutation process known as somatic hypermutation that allows the immune system to generate high-affinity antibodies. Somatic hypermutation preferentially affects Ig genes, relative to other genes, and this is important in preventing catastrophic level...
Article
RecQ family DNA helicases function in the maintenance of genome stability. Mice deficient in RecQL5, one of five RecQ helicases, show a cancer predisposition phenotype, suggesting that RecQL5 plays a tumor suppressor role. RecQL5 interacts with Rad51, a key factor in homologous recombination (HR), and displaces Rad51 from Rad51-single stranded DNA...
Article
Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes to support the generation of high affinity antibodies through affinity maturation. SHM is quite dangerous, having been shown to affect many non-Ig genes in germinal center and malignant B cells. Nonetheless, the reaction exhibits a strong preference for Ig loci ov...
Article
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Tie2 is a receptor tyrosine kinase that is essential for the development and maintenance of blood vessels through binding the soluble ligands angiopoietin 1 (Ang1) and 2 (Ang2). Ang1 is constitutively produced by perivascular cells and is protective of the adult vasculature. Ang2 plays an important role in blood vessel formation and is normally exp...
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Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of...
Data
(A) Representative cell-cycle distribution histograms of wt DT40 cells incubated for 10 d in the presence or absence of 4HT. (B) Growth curves of wt DT40 cells grown in the presence or absence of 4HT. Cells were maintained in the exponential phase of growth by daily dilution in fresh growth medium. (C) Repair kinetics of IR-induced DSBs in asynchro...
Data
(A) Western blot analysis of LIG3 protein in LIG3−/2loxPCdc9 cells after treatment with 4HT for the indicated periods of time, or when left untreated. A mouse monoclonal antibody against human LIG3 (clone 1F3) that recognizes the chicken LIG3 was used. GAPDH is a loading control. (B) Western blot analysis of LIG3 and RAD51 proteins in wt DT40 cells...
Data
(A) Growth kinetics of LIG3−/2loxPLIG4−/− cells and derivative clones 1, 3 and 7 expressing the mitochondrial version of hLIG1, LIG3−/2loxPLIG4−/−mts-hLIG1. Cells were maintained in the exponential phase of growth by daily dilution in fresh growth medium. (B) Human LIG1 mRNA level measured by real-time PCR in clones 1, 3 and 7 of LIG3−/2loxPLIG4−/−...
Data
(A) Growth kinetics of Lig3−/2loxPLig4−/−mts-hLig1 cells 5 d after 4HT treatment to convert to Lig3−/−Lig4−/−mts-hLig1 cells. The growth of Lig3−/2loxPLig4−/− cells immediately after treatment with 4HT is also shown for comparison. Cells were maintained in the exponential phase of growth by daily dilution in fresh growth medium. (B) Confirmation of...
Data
Full-text available
A summary of the key features of knockout, conditional knock-out and knock-in DT40 mutants generated and used in the present study. (PDF)
Article
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Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM a...
Article
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SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells sh...
Article
The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to...
Article
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In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-jo...
Article
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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 integrit...
Article
Somatic hypermutation (SHM) diversifies the rearranged immunoglobulin variable (V) region gene in B cells, contributing to affinity maturation of antibodies. It is believed that SHM is generated either by direct replication or by error-prone repair systems resolving V region DNA lesions caused directly or indirectly by cytidine deaminase AID. In ac...
Article
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Author Summary It remains an open question how AID-mediated gene diversification is targeted to the immunoglobulin loci. Here we define a cis-acting sequence, named DIVAC for diversification activator, which is required for hypermutation of the Ig light chain gene and sufficient to activate hypermutation at various non-Ig loci in the DT40 B cell li...
Data
(A) Targeting strategy of the GFP2 reporter into four different loci on chromosome 15. (B) Targeting strategy of the GFP2 reporter into the A-MYB, RAD52, BACH2, and BCL6 loci. The targeting strategies used for the insertions into the AID and the RMD1 loci were described previously [2],[29]. (1.87 MB EPS)
Data
Comparison of GFP gene expression levels analyzed by semi-quantitative RT-PCR of primary transfectants belonging to the stepwise deletions series of the ‘W’ fragment. (2.27 MB EPS)
Data
Green fluorescence decrease in individual primary transfectants. (A) GFP2 reporter in the vicinity of the IgL locus. (B) GFP2 reporter in non-Ig loci. (0.11 MB XLS)
Article
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Depending on the species and the developmental stage of B cells, activation-induced cytidine deaminase (AID) triggers immunoglobulin (Ig) gene diversification by gene conversion, hypermutation or switch recombination. The bursal B cell line DT40 usually diversifies its rearranged Ig light chain (IgL) gene by gene conversion, but disruption of the R...
Article
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Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied tho...
Article
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Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture...
Article
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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...
Article
DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol bet...
Article
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The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Sur...
Article
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The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by a...
Article
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Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in...
Article
Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, an...
Data
Full-text available
Gene Targeting Strategies and Screenings for Clones Having Undergone Targeted Integration Events Physical maps of the loci, the targeting vectors, and the targeted alleles are shown for PCNA (A), RAD18 (B), and REV1 (C), respectively. The position and orientation of the primers used for the screening by long-range PCR are indicated. The identificat...
Article
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A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-β locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-β gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-β chromatin was resistant to DNase...
Article
Full-text available
Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B...
Article
Genome projects have provided comprehensive gene catalogs and locus maps for many model organisms. Although sequence comparison and protein domain searches may suggest evolutionary conserved gene functions, genetic systems are still needed to determine the role of genes within living cells. Due to high ratios of targeted to random integration of tr...
Article
Chicken B cells develop their primary immunoglobulin (Ig) gene repertoire by pseudogene templated gene conversion within the bursa of Fabricius. The DT40 cell line is derived from bursal B cells and continues to diversify its rearranged Ig light chain in cell culture. Ig gene conversion of DT40 requires expression of the AID gene which was earlier...
Article
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The DT40 cell line is derived from ALV (avian leukosis virus)-transformed bursal B cell of chicken. This cell line is suspension cell, and can be cultured in cell culture bottles and in culture plates (6-, 24- and 96-well etc.). Optimal culture temperature for the DT40 is (39 degrees C-) 41 degrees C, because chicken has higher body temperature tha...
Article
Disruption of multiple genes and complementation of the phenotypes is restricted by the number of available drug-resistance genes. It is therefore highly desirable to recycle the drug-resistance genes using a site-specific recombination system like Cre/loxP. DT40(Cre1) cell line, which is transgenic with an inducible Cre recombinase (MerCreMer) is...
Article
Immunoglobulin (Ig) gene conversion and hypermutation rates can be quantified by FACS. The spontaneous DT40 variant, C118, and C118-derived DT40 mutants can be used to analyze gene conversion phenotype. The C118 has a frameshift in its rearranged V segment. When this frameshift mutation is repaired by pseudogene-templated gene conversion, sIgM(-) D...
Article
After the stable transfection of the knockout vector, the transfectants successful for targeted integration can be screened by PCR. A primer located upstream of the 5' targeting arm of the construct can be used for this PCR together with a primer from the resistance marker. PCR screening is more advantageous than Southern screening not only for spe...
Article
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In order to understand the function of genes, transfection can be used as a method in which artificially prepared knockout and expression constructs are being introduced into cell lines. Since many genes are essential for embryonic development and a homozygous deletion results in non-viable embryos, gene disruption in a cell line by using transfect...
Article
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...
Article
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A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searc...
Article
The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell-specific Ig-beta gene and in maintenance of active chromatin state in the Ig-beta locus were examined. A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocyte-derived DT40 cells. Deletion of three DHSs located bet...
Article
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A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekee...
Article
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Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event f...
Article
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The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of...
Article
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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...
Article
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Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated 2-fold in chicken DT40 ce...