APTX acts in DNA double-strand break repair in a manner distinct from XRCC4.

Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear.

Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.

The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability.

The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability.

Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites.

USP42 enhances homologous recombination repair by promoting R-loop resolution with a DNA-RNA helicase DHX9.

The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection.

Bacterial SOS Genes Promote Mouse Tumors by Activating Oncogenes via a miR-145 Sponge.

The mechanism of cancer induction involves an aberrant expression of oncogenes whose functions can be controlled by RNAi with miRNA. Even foreign bacterial RNA may interfere with the expression of oncogenes. Here we show that bacterial plasmid and its genomic homolog carrying homologies that match the mouse anti-miR-145, sequestered the miR-145 function in mouse BALB 3T3 cells in a tetracycline (Tet)-inducible manner, activated oncogene and its downstream and further enhanced microcolony formation and cellular transformation as well as the short fragments of the bacterial gene containing the anti-miR-145 sequence.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS.

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear.

PARI Regulates Stalled Replication Fork Processing To Maintain Genome Stability upon Replication Stress in Mice.

DNA replication is frequently perturbed by intrinsic, as well as extrinsic, genotoxic stress. At damaged forks, DNA replication and repair activities require proper coordination to maintain genome integrity. We show here that PARI antirecombinase plays an essential role in modulating the initial response to replication stress in mice.

Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia.

The WD40-containing E3 ubiquitin ligase RFWD3 has been recently linked to the repair of DNA damage by homologous recombination (HR). Here we have shown that an RFWD3 mutation within the WD40 domain is connected to the genetic disease Fanconi anemia (FA). An individual presented with congenital abnormalities characteristic of FA.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3.

Activation of the FA pathway mediated by phosphorylation and ubiquitination.

Fanconi anemia (FA) is a devastating hereditary condition that impacts genome integrity, leading to clinical features such as skeletal and visceral organ malformations, attrition of bone marrow stem cells, and carcinogenesis. At least 21 proteins, when absent or defective, have been implicated in this disorder, and they together constitute the FA pathway, which functions in detection and repair of, and tolerance to, endogenous DNA damage. The damage primarily handled by the FA pathway has been assumed to be related to DNA interstrand crosslinks (ICLs).

Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament.

Defective FANCI binding by a fanconi anemia-related FANCD2 mutant.

FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution.

The Trp53-Trp53inp1-Tnfrsf10b pathway regulates the radiation response of mouse spermatogonial stem cells.

Germ cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation.

FANCD2 binds CtIP and regulates DNA-end resection during DNA interstrand crosslink repair.

The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2.

Tumor suppressor RecQL5 controls recombination induced by DNA crosslinking agents.

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 (ssDNA) filaments in vitro.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans.

Establishment of the DNA repair-defective mutants in DT40 cells.

The chicken B cell line DT40 has been widely used as a model system for reverse genetics studies in higher eukaryotes, because of its advantages including efficient gene targeting and ease of chromosome manipulation. Although the genetic approach using the RNA interference technique has become the standard method particularly in human cells, DT40 still remains a powerful tool to investigate the regulation and function of genes and proteins in a vertebrate system, because of feasibility of easy, rapid, and clear genetic experiments. The use of DT40 cells for DNA repair research has several advantages.

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood.