Meiotic DNA repair in Arabidopsis thaliana

Meiose ist eine spezielle Form der Zellteilung die den diploiden Chromosomensatz auf einen haploiden reduziert. Zwei Kernteilungen folgen einer prämeiotischen DNA Replikation, so dass schließlich vier haploide Tochterzellen vorliegen. Während in der ersten meiotischen Teilung die Trennung der homologen Chromosomen erfolgt, werden in der zweiten meiotischen Teilung die Schwesterchromatiden getrennt. In der Meiose kommt es zum reziproken Austausch genetischer Information, ein Prozess der homologe Rekombination genannt wird. Spo11, ein verwandtes Protein der archeabakteriellen Topoisomeraseuntereinheit Top6A, leitet die HR durch die kontrollierte Einfügung von Doppelstrangbrüchen ein. In der Bäckerhefe Saccharomyces cerevisiae sind die Proteine Mre11, Rad50, Xrs2 und Com1/Sae2 essentiell um diese Doppelstrangbrüche zu prozessieren und in weitere Folge, deren Reparatur zu ermöglichen. Die Arabidopsis thaliana Atcom1-1 Mutante ist steril. Darüber hinaus reichert sich AtSPO11-1 während der meiotischen Prophase I an. Das Fehlen von AtRAD51 in der Atcom1-1 Mutante deutet auf nicht reparierte Doppelstrangbrüche hin. Die für Atcom1-1 typische DNA Fragmentierung kann durch Mutation von AtSPO11-1 unterdrückt werden. In dieser Studie konnten wir interessanterweise eine Interaktion von AtCOM1 mit AtNBS1, einem Protein der DNA Reparaturmaschinerie, nachweisen. Nach erfolgter Prozessierung der Doppelstrangbrüche dient die einzelsträngige DNA (ssDNA) als Sonde zum Auffinden des homologen Partnerchromosoms. Für diesen entscheidenden Schritt der DNA Homologiesuche sind mehrere Proteine nötig. Unter anderem wurde Mnd1 als eines für den Strangaustausch essentielles Protein identifiziert. Das homologe Protein in Arabidopsis, AtMND1, ist, gemeinsam mit AHP2, dem Homologen von Hop2 der Hefe, wichtig für die Meiose. Weitere Proteine, wie die RecA Homologen DMC1, RAD51 und XRCC3 sind essentiell für den korrekten Austausch des genetischen Materials. Über das Zusammenspiel dieser Proteine ist bis jetzt wenig bekannt. Das Fehlen von AtMND1 verursacht schwere DNA Fragmentierung. Eine Akkumulierung von AtRAD51, wie auch von AtDMC1 kann in der Atmnd1 Mutante beobachtet werden. Dies ist auf nicht reparierte DSB zurückzuführen. Immunolokalisationsstudien zeigen die funktionellen Unterschiede von AtRAD51 and AtXRCC3 während der Meiose auf. AtXRCC3 ist für die Nukleoproteinfilamentbildung des AtDMC1 Proteins in einer Atmnd1 Mutante entbehrlich, während AtXRCC3 unverzichtbar für eine effiziente Anlagerung von AtRAD51 an die prozessierten ssDNA Enden ist.Meiosis is a specialized nuclear division characteristic for sexually reproducing eukaryotes. Each diploid progenitor generates four genetically different haploid cells proceeding through two successive nuclear divisions that follow a single round of genome replication. This process relies on meiotic homologous recombination (HR) that establishes a physical connection between pairs of homologs and allows the correct alignment of bivalents. Moreover, genetic diversity is generated by the exchange of DNA sequences between maternal and paternal chromosomes. Homologous recombination is initiated by programmed DNA double strand breaks (DSBs) catalyzed by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Mre11, Rad50, Xrs2 and Com1/Sae2 are essential to process these DSBs. Arabidopsis thaliana Atcom1-1 mutants are sterile, accumulating AtSPO11-1 during meiotic prophase and failing to form AtRAD51 foci, indicative for un-processed DSBs. Furthermore, DNA fragmentation seen in Atcom1-1 mutants is suppressed in the absence of AtSPO11-1, pointing to a defect in DSB repair. In accordance with data in other organisms, we found that AtCOM1 interacts with AtNBS1, a protein which is involved in the early steps of DNA repair. After processing of DSBs, a single stranded DNA molecule recognizes and invades the homologous sequence. Many proteins are involved in this crucial step of homology search. Among others Mnd1 has been identified as one of the key players in the strand invasion process. The Arabidopsis homologue, AtMND1, is essential for male and female meiosis. Furthermore, other proteins involved in meiotic recombination can be found in Arabidopsis thaliana, for instance the RecA related proteins DMC1, RAD51 and XRCC3. Few is known about the interplay between these proteins during meiosis. AtMND1 promotes the strand invasion process together with AHP2, the Arabidopsis protein closely related to budding yeasts Hop2. In the absence of AtMND1, severe chromosome fragmentation is observed, depending on the presence of AtSPO11-1. Moreover AtRAD51 as well as AtDMC1 foci, have been observed to accumulate on cytological preparations of meiotic cells. They demonstrate that DNA breaks remain unrepaired. Furthermore, immunolocalization studies provide insight into the functional differences of AtRAD51 and AtXRCC3 during meiosis, by first demonstrating that AtXRCC3 is dispensable for AtDMC1 nucleoproteinfilament formation in an Atmnd1 mutant background, and second that AtXRCC3 is indispensable for efficient loading of AtRAD51

The leader region of Laminin B1 mRNA confers cap-independent translation

Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5′-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5′-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes

The Interplay of RecA-related Proteins and the MND1–HOP2 Complex during Meiosis in Arabidopsis thaliana

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1–Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1–AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3

LamB1 translation after heat shock or intervention with ribosome scanning through 2A protease-dependent cleavage of eIF4G

<p><b>Copyright information:</b></p><p>Taken from "The leader region of Laminin B1 mRNA confers cap-independent translation"</p><p></p><p>Nucleic Acids Research 2007;35(8):2473-2482.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885646.</p><p>© 2007 The Author(s)</p> () LamB1 expression in MIM-R cells 4, 6 and 8 h after heat shock as detected by western blotting. () Firefly luciferase assay of MIM-R hepatocytes transfected either with pR-EMCV-F or pR-Lam-F bicistronic plasmids. Cells were exposed to heat shock 12 h post-transfection. 48 hours after transfection, Firefly luciferase activity was determined and normalized to the RNA level after reverse transcription and quantitation of cDNA. () Western blot analysis of MIM-R cells showing the cleavage of eIF4GI/II. MIM-R cells were transfected with wild-type 2A protease expressing plasmid (p2Awt) and lysed at the indicated times. () Firefly luciferase assay of MIM-R hepatocytes co-transfected with pR-F and either p2Amut or p2Awt. () Firefly luciferase assay of MIM-R cells co-transfected either with pR-EMCV-F or pR-Lam-F and p2Amut or p2Awt, respectively. Cells were lysed 48 h post-transfection and the Firefly luciferase activity was normalized to the RNA level after reverse transcription and quantitation of cDNA

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination

Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination