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. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway

Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (τ), quantitative trait locus (QTL) analysis was performed using F2 mice obtained from an F1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For τ, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes

DNA ソンショウ ニ ヨッテ ユウドウサレル RFC フクゴウタイ サブユニット RFC2 ノ ユビキチンカ

京都大学0048新制・課程博士博士(医学)甲第13735号医博第3250号新制||医||968(附属図書館)UT51-2008-C651京都大学大学院医学研究科生理系専攻(主査)教授 武田 俊一, 教授 髙田 穣, 教授 松本 智裕学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

FANCD2 Binds CtIP and Regulates DNA-End Resection during DNA Interstrand Crosslink Repair

小児の遺伝性疾患「ファンコニ貧血」病態の完全解明への一歩 ～キー分子FANCD2に会合するCtIPタンパク質の同定～. 京都大学プレスリリース. 2014-05-02.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. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMCinduced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair

FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress

R‐loops, which consist of DNA : RNA hybrids and displaced single‐strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R‐loops. In this study, we found that FANCD2 suppresses R‐loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R‐loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription–replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R‐loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress

Alternative Invasion Mechanisms and Host Immune Response to Plasmodium vivax Malaria: Trends and Future Directions

Plasmodium vivax malaria is a neglected tropical disease, despite being more geographically widespread than any other form of malaria. The documentation of P. vivax infections in different parts of Africa where Duffy-negative individuals are predominant suggested that there are alternative pathways for P. vivax to invade human erythrocytes. Duffy-negative individuals may be just as fit as Duffy-positive individuals and are no longer resistant to P.vivax malaria. In this review, we describe the complexity of P. vivax malaria, characterize pathogenesis and candidate invasion genes of P. vivax, and host immune responses to P. vivax infections. We provide a comprehensive review on parasite ligands in several Plasmodium species that further justify candidate genes in P. vivax. We also summarize previous genomic and transcriptomic studies related to the identification of ligand and receptor proteins in P. vivax erythrocyte invasion. Finally, we identify topics that remain unclear and propose future studies that will greatly contribute to our knowledge of P. vivax

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

<div><p>DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the <i>Rev3l</i> gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that <i>Rev3l</i> cDNA rescues the genomic instability and DNA damage sensitivity of <i>Rev3l</i>-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of <i>REV3L</i> could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity <i>in vivo</i>, a <i>Rev3l</i> knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to <i>Rev3l</i>-null fibroblasts. We tested whether the severe <i>Rev3l</i>^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, <i>Rev3l</i>^-/-<i>Polh</i>^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as <i>Rev3l</i>^-/-<i>Polh</i>^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.</p></div

Knock-in mice and MEFs expressing active site mutant <i>Rev3l</i> have knockout phenotypes.

<p>(A) Diagram of the mouse <i>Rev3l</i> ASM knock-in allele, with the wild-type (WT) locus shown at the top. Green rectangles indicate <i>Rev3l</i> coding sequences and the gray line represents chromosomal sequence. In the middle diagram, FRT sites are represented by double red triangles, loxP sites by blue triangles and lox511 sites by yellow triangles. The targeted exon 27 (starred) carries D2773A and D2775A point mutations and is inserted in an inverted orientation between wild-type exons 26 and 27. Splicing donor and acceptor sites flanking wild-type and ASM exon 27 are kept intact. The knock-in was produced by a Cre-dependent genetic switch. First, the neomycin positive selection cassette (neo) was excised by breeding with C57BL/6 Flp deleter mice. A subsequent cross with Cre-expressing mice led to excision of the wild-type exon 27 and inversion of ASM mutant exon 27 into the functional orientation. In the constitutive ASM knock-in locus shown in the lower diagram, the D2773A/D2775A <i>Rev3l</i> gene is expressed under the control of the endogenous <i>Rev3l</i> promoter and wild-type <i>Rev3l</i> exon 27 is absent from the locus. Heterozygous ASM knock-in mice (<i>Rev3L</i>^+/M) were then used for breeding. (B) Example of Southern blot analysis of (left) the inducible knock-in locus (<i>neo</i>⁺) and (right) the constitutive ASM knock-in locus. Genomic DNA of the tested animals was compared with C57BL/6 wild-type genomic DNA (WT). <i>Eco</i>RV digested DNA was blotted on a nylon membrane and hybridized with the external 3’ probe with the position shown at the top of part A. Restriction fragments of 15 kb, 11.5 kb and 9.5 kb were observed for the wild-type, inducible knock-in locus (<i>neo</i>⁺) and constitutive ASM knock-in locus, respectively. Genomic DNA was further analyzed extensively and confirmed by specific PCR assays and complete DNA sequencing as described in the Materials and Methods. (C) Genotypes of mouse pups produced by breeding parental <i>Rev3l</i>^+/M mice. (D) Growth of <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ cells. These cells were produced by addition of AdCre to <i>Rev3l</i>^M/lox or <i>Rev3l</i>^+/lox MEFs, deleting the floxed allele of <i>Rev3l</i>. (E) Survival of <i>Rev3l</i>^+/lox, <i>Rev3l</i>^M/lox, <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ primary MEFs 120 hr after addition of cisplatin, as measured by ATP content. (F) The MEFs as in (E) were stained with DAPI, and for 53BP1 and γ-H2AX by immunofluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005759#pgen.1005759.g002" target="_blank">Fig 2</a> to detect foci of DNA double-strand breaks. The quantification shows the percentage of cells with >2 53BP1 and γ-H2AX foci in <i>Rev3l</i>^+/lox, <i>Rev3l</i>^M/lox, <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ primary MEFs 9 days after AdCre treatment. (*) p < 0.01. Data represent mean ± SEM.</p