Homologous Recombination Is Stimulated by a Decrease in dUTPase in Arabidopsis

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations

Recherche et étude de fonctions impliquées dans le métabolisme de l'ADN chez Arabidopsis thaliana

Afin d identifier de nouvelles fonctions potentiellement impliquées dans le métabolisme de l ADN chez Arabidopsis, nous avons utilisé un crible bactérien décrit par Perkins et al. (1999). Nous avons isolé 100 candidats capables d induire la réponse SOS chez E. coli. La moitié, au moins, de ces fonctions pourrait être impliquée dans le métabolisme de l ADN chez Arabidopsis, alors que 23 autres correspondent à des fonctions inconnues. Pour approcher in planta la fonction des candidats isolés, nous avons étudié leur expression (en utilisant des macroarrays), recherché des interactions avec des protéines connues impliquées dans la réparation/recombinaison de l ADN et étudié, pour certains d entre-eux, leur fonction en utilisant l interférence par ARN (RNAi). La recherche d interactions à grande échelle et l extinction de gènes par RNAi ont été calibrés avec les gènes impliqués dans la recombinaison homologue (RH) de l ADN. Ces approches ont permis de montrer que Brca2 (dont les mutations prédisposent les mammifères au cancer) est essentiellle à la méiose chez Arabidopsis et de proposer un rôle pour Brca2 à la méiose : Brca2 dirige la réparation des cassures double-brin méiotiques de l ADN (introduites par Spo11) vers la RH. Son rôle réside probablement dans ses interactions avec les recombinases Rad51 et Dmc1, l interaction entre les protéines Brca2 et Dmc1 n ayant jamais été décrite avant. En l absence de Brca2, ou de l absence simultanée de Rad51 et Dmc1, la RH est éliminée, et la réparation des cassures double-brin de l ADN se ferait par des mécanismes de recombinaison illégitime, conduisant à la formation de structures chromosomiques aberrantes.In the aim to identify new functions putatively involved in DNA metabolism in Arabidopis, we used a bacterial screen described by Perkins et al. (1999). We isolated 100 candidates that can trigger the SOS reponse in E. coli. Half of them could be involved in DNA metabolism, while 23 others correspond to unknown functions. To investigate their in planta function, we examined their expression (using macroarrays), searched for interactions with known proteins involved in DNA repair/recombination, and studied, for a few of them, their function using RNA interference (RNAi).Search for interactions and gene silencing via RNAi were calibrated with genes involved in homologous recombination. These approaches allowed us to show that Brca2 (whose mutations predispose mammals to cancer) is essential at meiosis in Arabidopsis, and to propose a role for Brca2 at meiosis: Brca2 directs the repair of meiotic DNA double-strand break (introduced by Spo11) towards homologous recombination. Brca2 s role probably resides in its interaction with the recombinases Rad51 and Dmc1 (the interaction between Brca2 and Dmc1 had never been described before). In the absence of Brca2, or in the absence of both Rad51 and Dmc1, homologous recombination is abolished and meiotic DNA double-strand breaks seem to be repaired by illegitimate recombination, leading to the formation of abnormal chromosomal structures.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Interaction between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I)

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The ‘brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context

BRCA2 peptides functional in HR.

<p>A. Micro-BRCA2 domain structure. Micro-BRCA2 contains three domains – the PALB2 interaction site, BRC1–2, and the Cter, whereas zippo-BRCA2 is deleted for the BRC repeats. B. Western blot analysis showing expression of the indicated BRCA2 peptides. C. Comparison of HR activity of various BRCA2 peptides. Micro-BRCA2 is more active than mini-BRCA2 (<i>P</i> = 0.0024), whereas zippo-BRCA2 is not active. D. Summary of BRCA2 peptides and their relative HR activity. E. Schematic of peptides active in HR. While full-length BRCA2 has optimal HR activity, BRCA2 peptides can be derived which have substantial activity. Those that bind PALB2, such as micro-BRCA2, are active even with a deleted DNA binding domain (DBD). In the absence of PALB2 binding, an intact DBD is critical for HR activity. BRC fusion to RPA, however, can bypass the requirement for the DBD.</p

HR proficiency of mini-BRCA2 peptides.

<p>A. Human BRCA2 domain structure. BRCA2 binds RAD51 at the eight BRC repeats in the central region of the protein and at a distinct site in the C-terminus (Cter). In addition, BRCA2 binds PALB2 at the N terminus and has a conserved DSS1 and DNA-binding domain (DBD). B. Mini-BRCA2 domain structures. Peptides contain the indicated BRCA2 domains as well as a nuclear localization signal (nls) at the N terminus and a FLAG epitope tag at the C terminus. C. DR-GFP. The DR-GFP reporter consists of two defective <i>GFP</i> genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002409#pgen.1002409-Pierce1" target="_blank">[29]</a>. Expression of I-SceI endonuclease results in a double-strand break (DSB) at the I-SceI site in the <i>SceGFP</i> gene which can be repaired using the homologous sequence in the <i>iGFP</i> gene to generate GFP positive cells which are quantified by flow cytometry. D. BRCA2-complemented V-C8 cells have substantially higher DSB-induced HR than uncomplemented cells. <i>P</i> = 0.0002, two-tailed unpaired t test. A bacterial artificial chromosome (BAC) (clone RP11-777I19; BACPAC Resource Center at the Children's Hospital Oakland Research Institute in Oakland, California) which contains human <i>BRCA2 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002409#pgen.1002409-Sharan2" target="_blank">[53]</a> was stably introduced into V-C8 DR-GFP cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002409#pgen.1002409-Saeki1" target="_blank">[12]</a> using a linked neomycin resistance gene for selection. BRCA2 expression was verified by Western blot analysis (not shown). E. Mini-BRCA2s containing the Cter partially correct the HR defect of <i>Brca2</i>-deficient V-C8 hamster cells. Mini-BRCA2s that have activity relative to no mini-BRCA2 are BRC3-DBD-Cter (<i>P</i><0.05), and BRC1–2-DBD-Cter and BRC1–4-DBD-Cter (<i>P</i>≤0.001; unpaired <i>t</i> test). All peptides (red asterisks) are of the expected sizes. BRC1–4-DBD-Cter corrects the HR defect as well or better than BRC1–2-DBD-Cter despite being poorly detected. F. Comparison of HR activity at different levels of BRCA2 peptide expression. The BRC1–2-DBD-Cter expression vector was transfected at the usual amount (50 µg) or at a reduced amount (10 µg). HR levels were similar in both cases (<i>P</i> = 0.82). G. Mini-BRCA2s with multiple BRC repeats are more active in HR than those with single BRC repeats. Relative to BRC1–2-DBD-Cter, <i>P</i>≤0.0001 for single repeat mini-BRCA2s, except for BRC2-DBD-Cter where <i>P</i> = 0.003. All mini-BRCA2s have significant activity relative to no mini-BRCA2 (<i>P</i>≤0.005), except BRCΔ3-DBD-Cter (<i>P</i> = 0.248).</p