The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis

<p>Abstract</p> <p>Background</p> <p>Meiotic recombination in eukaryotes requires two homologues of the <it>E. coli </it>RecA proteins: Rad51 and Dmc1. Both proteins play important roles in the binding of single stranded DNA, homology search, strand invasion and strand exchange. Meiotic recombination has been well studied in Arabidopsis, rice, maize and the orthologues of <it>RAD51 </it>and <it>DMC1 </it>have been characterized. However genetic analysis of the <it>RAD51 </it>and <it>DMC1 </it>genes in bread wheat has been hampered due to the absence of complete sequence information and because of the existence of multiple copies of each gene in the hexaploid wheat genome.</p> <p>Findings</p> <p>In this study we have identified that <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of <it>TaRAD51 </it>and <it>TaDMC1 </it>and those of other eukaryotes reveal a high degree of evolutionary conservation. Despite the high degree of sequence conservation at the nucleotide level, genome-specific primers for cDNAs of <it>TaRAD51 </it>and <it>TaDMC1 </it>were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the <it>TaRAD51 </it>and <it>TaDMC1 </it>cDNA homoeologues was largely restricted to meiotic tissue, with elevated levels observed during the stages of prophase I when meiotic recombination occurs. All three homoeologues of both strand-exchange proteins (<it>TaRAD51 </it>and <it>TaDMC1</it>) are expressed in wheat.</p> <p>Conclusions</p> <p>Bread wheat contains three expressed copies of each of the <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues. While differences were detected between the three cDNA homoeologues of <it>TaRAD51 </it>as well as the three homoeologues of <it>TaDMC1</it>, it is unlikely that the predicted amino acid substitutions would have an effect on the protein structure, based on our three-dimensional structure prediction analyses. There are differences in the levels of expression of the three homoeologues of <it>TaRAD51 </it>and <it>TaDMC1 </it>as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.</p

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity

Identification of Nucleases and Phosphatases by Direct Biochemical Screen of the Saccharomyces cerevisiae Proteome

The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His6, an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins

Chromatin organization revealed by nanostructure of irradiation induced gamma H2AX, 53BP1 and Rad51 foci

The spatial distribution of DSB repair factors gamma H2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and gamma H2AX form IRIF with same mean size of (540 +/- 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by gamma H2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 +/- 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 +/- 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened

Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p>In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. <it>RAD52 </it>epistasis group genes involved in recombinational DNA repair, including <it>mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 </it>and <it>rad59 </it>genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in <it>Entamoeba histolytica </it>the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.</p> <p>Results</p> <p>In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote <it>E. histolytica </it>using UV-C (150 J/m²) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In <it>E. histolytica </it>genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the <it>E. histolytica </it>RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear <it>foci</it>-like structures in <it>E. histolytica </it>trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands <it>in vitro</it>.</p> <p>Conclusion</p> <p><it>E. histolytica </it>genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear <it>foci</it>-like structures. Functional assays confirmed that EhRAD51 is a <it>bonafide </it>recombinase. These data provided the first insights about the potential roles of the <it>E. histolytica </it>RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.</p

Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics