Frequency Of Gene Conversion Research Articles

Measuring Meiotic Recombination Frequency in Schizosaccharomyces pombe Using an Engineered Genetic Interval.

The fission yeast Schizosaccharomyces pombe has been used to elucidate meiotic recombination mechanisms for decades. Alongside the budding yeast Saccharomyces cerevisiae, research employing fission yeast has been instrumental in advancing our knowledge of double-stranded DNA break (DSB) formation and repair during meiosis. Genetic recombination assays are the workhorses of gene conversion and crossover frequency analysis; these have been employed to investigate cis and trans determinants of meiotic recombination. Here, I describe meiotic recombination assays engineered by the introduction of nutritional markers up- and downstream of the ade6 and ade7 genes. These particular setups enable a comprehensive assessment of reproductive success in a single assay because spore viability and the frequency of gene conversion, crossovers, and crossovers associated with gene conversion events are simultaneously measured.

Molecular evolution of the hemoglobin gene family across vertebrates

Adaptation to various altitudes and oxygen levels is a major aspect of vertebrate evolution. Hemoglobin is an erythrocyte protein belonging to the globin superfamily, and the α-, β-globin genes of jawed vertebrates encode tetrameric ((α2β2) hemoglobin, which contributes to aerobic metabolism by delivering oxygen from the respiratory exchange surfaces into cells. However, there are various gaps in knowledge regarding hemoglobin gene evolution, including patterns in cartilaginous fish and the roles of gene conversion in various taxa. Hence, we evaluated the evolutionary history of the vertebrate hemoglobin gene family by analyses of 97 species representing all classes of vertebrates. By genome-wide analyses, we extracted 879 hemoglobin sequences. Members of the hemoglobin gene family were conserved in birds and reptiles but variable in mammals, amphibians, and teleosts. Gene motifs, structures, and synteny were relatively well-conserved among vertebrates. Our results revealed that purifying selection contributed substantially to the evolution of all vertebrate hemoglobin genes, with mean dN/dS (ω) values ranging from 0.057 in teleosts to 0.359 in reptiles. In general, after the fish-specific genome duplication, the teleost hemoglobin genes showed variation in rates of evolution, and the β-globin genes showed relatively high ω values after a gene transposition event in amniotes. We also observed that the frequency of gene conversion was high in amniotes, with fewer hemoglobin genes and higher rates of evolution. Collectively, our findings provide detail insight into complex evolutionary processes shaping the vertebrate hemoglobin gene family, involving gene duplication, gene loss, purifying selection, and gene conversion.

Unexpectedly high mutation rate of cyp11b1 compared to cyp21a2 in randomly-selected turkish women: a large screening study.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders resulting from enzyme deficiencies associated with steroidogenesis. The clinical presentation of non-classic CAH (NCAH) in females is often indistinguishable from other hyperandrogenic disorders like polycystic ovary syndrome (PCOS). The data on the prevalence of NCAH in unselected women in the literature is scanty. The research aimed to evaluate the prevalence of NCAH, carrier frequencies, and the correlation between clinical symptoms and genotype in Turkish women. The study group comprised two hundred and seventy randomly-selected unrelated asymptomatic women of reproductive age (18-45). Subjects were recruited from female blood donors. All volunteers underwent clinical examination and hormone measurements. The protein-encoding exons and exon-intron boundaries of the CYP21A2, CYP11B1, HSD3β2 and CYP21A2 promoter were sequenced by direct DNA sequencing. After genotyping, seven (2.2%) individuals were diagnosed with NCAH. The heterozygous carrier frequencies of CYP21A2, CYP21A2 promoter, CYP11B1, and HSD3β2 genes with 34, 34, 41, and 1 pathologic mutation were determined at 12.6%, 12.6%, 15.2%, and 0.37% of volunteers, respectively. Gene-conversion (GC) frequencies between CYP21A2/CYP21A1P and CYP11B1/CYP11B2 were determined as 10.4% and 14.8%, respectively. Despite GC-derived higher mutation frequency determined in the CYP11B1 gene, the reason for the low frequency of NCAH due to 11OHD compared to 21OHD might be that gene-conversion arises with active CYP11B2 rather than an inactive pseudogene. HSD3β1 exhibits high homology with HSD3β2 located on the same chromosome; remarkably, it demonstrates low heterozygosity and no GC, most probably the outcome of a tissue-specific expression pattern.

Conversion between 100-million-year-old duplicated genes contributes to rice subspecies divergence

BackgroundDuplicated gene pairs produced by ancient polyploidy maintain high sequence similarity over a long period of time and may result from illegitimate recombination between homeologous chromosomes. The genomes of Asian cultivated rice Oryza sativa ssp. indica (XI) and Oryza sativa ssp. japonica (GJ) have recently been updated, providing new opportunities for investigating ongoing gene conversion events and their impact on genome evolution.ResultsUsing comparative genomics and phylogenetic analyses, we evaluated gene conversion rates between duplicated genes produced by polyploidization 100 million years ago (mya) in GJ and XI. At least 5.19–5.77% of genes duplicated across the three rice genomes were affected by whole-gene conversion after the divergence of GJ and XI at ~ 0.4 mya, with more (7.77–9.53%) showing conversion of only portions of genes. Independently converted duplicates surviving in the genomes of different subspecies often use the same donor genes. The ongoing gene conversion frequency was higher near chromosome termini, with a single pair of homoeologous chromosomes, 11 and 12, in each rice genome being most affected. Notably, ongoing gene conversion has maintained similarity between very ancient duplicates, provided opportunities for further gene conversion, and accelerated rice divergence. Chromosome rearrangements after polyploidization are associated with ongoing gene conversion events, and they directly restrict recombination and inhibit duplicated gene conversion between homeologous regions. Furthermore, we found that the converted genes tended to have more similar expression patterns than nonconverted duplicates. Gene conversion affects biological functions associated with multiple genes, such as catalytic activity, implying opportunities for interaction among members of large gene families, such as NBS-LRR disease-resistance genes, contributing to the occurrence of the gene conversion.ConclusionDuplicated genes in rice subspecies generated by grass polyploidization ~ 100 mya remain affected by gene conversion at high frequency, with important implications for the divergence of rice subspecies.

Characterization of Non-selected Intermolecular Gene Conversion in the Polyploid Haloarchaeon Haloferax volcanii.

Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.

Approximating the Coalescent Under Facultative Sex.

Genome studies of facultative sexual species, which can either reproduce sexually or asexually, are providing insight into the evolutionary consequences of mixed reproductive modes. It is currently unclear to what extent the evolutionary history of facultative sexuals' genomes can be approximated by the standard coalescent, and if a coalescent effective population size Ne exists. Here, I determine if and when these approximations can be made. When sex is frequent (occurring at a frequency much greater than 1/N per reproduction per generation, for N the actual population size), the underlying genealogy can be approximated by the standard coalescent, with a coalescent Ne≈N. When sex is very rare (at frequency much lower than 1/N), approximations for the pairwise coalescent time can be obtained, which is strongly influenced by the frequencies of sex and mitotic gene conversion, rather than N. However, these terms do not translate into a coalescent Ne. These results are used to discuss the best sampling strategies for investigating the evolutionary history of facultative sexual species.

Analysis of the recombination landscape of hexaploid bread wheat reveals genes controlling recombination and gene conversion frequency

BackgroundSequence exchange between homologous chromosomes through crossing over and gene conversion is highly conserved among eukaryotes, contributing to genome stability and genetic diversity. A lack of recombination limits breeding efforts in crops; therefore, increasing recombination rates can reduce linkage drag and generate new genetic combinations.ResultsWe use computational analysis of 13 recombinant inbred mapping populations to assess crossover and gene conversion frequency in the hexaploid genome of wheat (Triticum aestivum). We observe that high-frequency crossover sites are shared between populations and that closely related parents lead to populations with more similar crossover patterns. We demonstrate that gene conversion is more prevalent and covers more of the genome in wheat than in other plants, making it a critical process in the generation of new haplotypes, particularly in centromeric regions where crossovers are rare. We identify quantitative trait loci for altered gene conversion and crossover frequency and confirm functionality for a novel RecQ helicase gene that belongs to an ancient clade that is missing in some plant lineages including Arabidopsis.ConclusionsThis is the first gene to be demonstrated to be involved in gene conversion in wheat. Harnessing the RecQ helicase has the potential to break linkage drag utilizing widespread gene conversions.

On the representation of probability vector with special diffusion operator using the mutation and gene conversion rate

We will deal with an $n$ locus model in which mutation and gene conversion are taken into consideration. Also random partitions of the number $n$ determined by chromosomes with $n$ loci should be investigated. The diffusion process describes the time evolution of distributions of the random partitions. In this paper, we find the probability of distribution of the diffusion process with special diffusion operator $L_1$ and we show that the average probability of genes at different loci on one chromosome can be described by the rate of gene frequency of mutation and gene conversion.

Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation.

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.

Prevention of Transcriptional γ-globin Gene Silencing by Inducing The Hereditary Persistence of Fetal Hemoglobin Point Mutation Using Chimeraplast-Mediated Gene Targeting.

Objective Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. ConclusionOur results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

Gene Conversion Facilitates Adaptive Evolution on Rugged Fitness Landscapes.

Gene conversion is a ubiquitous phenomenon that leads to the exchange of genetic information between homologous DNA regions and maintains coevolving multi-gene families in most prokaryotic and eukaryotic organisms. In this paper, we study its implications for the evolution of a single functional gene with a silenced duplicate, using two different models of evolution on rugged fitness landscapes. Our analytical and numerical results show that, by helping to circumvent valleys of low fitness, gene conversion with a passive duplicate gene can cause a significant speedup of adaptation, which depends nontrivially on the frequency of gene conversion and the structure of the landscape. We find that stochastic effects due to finite population sizes further increase the likelihood of exploiting this evolutionary pathway. A universal feature appearing in both deterministic and stochastic analysis of our models is the existence of an optimal gene conversion rate, which maximizes the speed of adaptation. Our results reveal the potential for duplicate genes to act as a "scratch paper" that frees evolution from being limited to strictly beneficial mutations in strongly selective environments.

Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 μg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 μg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

Chemical Composition and Pharmacological Effects of Geopropolis Produced by Melipona quadrifasciata anthidioides.

Stingless bees produce geopropolis, which is popularly described for its medicinal properties, but for which few scientific studies have demonstrated pharmacological effects. The objective of this study was to investigate the chemical composition of the geopropolis of Melipona quadrifasciata anthidioides and to evaluate its antioxidant, antimutagenic, anti-inflammatory, and antimicrobial activities. The composition of the hydroethanolic extract of geopropolis (HEG) included di- and trigalloyl and phenylpropanyl heteroside derivatives, flavanones, diterpenes, and triterpenes. HEG showed antioxidant action via the direct capture of free radicals and by inhibiting the levels of oxidative hemolysis and malondialdehyde in human erythrocytes under oxidative stress. HEG also reduced the frequency of gene conversion and the number of mutant colonies of S. cerevisiae. The anti-inflammatory action of HEG was demonstrated by the inhibition of hyaluronidase enzyme activity. In addition, HEG induced cell death in all evaluated gram-positive bacteria, gram-negative bacteria, and yeasts, including clinical isolates with antimicrobial drug resistance. Collectively, these results demonstrate the potential of M. q. anthidioides geopropolis for the prevention and treatment of various diseases related to oxidative stress, mutagenesis, inflammatory processes, and microbial infections.

PCNA SUMOylation protects against PCNA polyubiquitination-mediated, Rad59-dependent, spontaneous, intrachromosomal gene conversion

Homologous recombination is crucial in both the maintenance of genome stability and the generation of genetic diversity. Recently, multiple aspects of the recombination machinery functioning at arrested DNA replication forks have been established, yet the roles of diverse modifications of PCNA, the key platform organizing the replication complex, in intrachromosomal recombination have not been comprehensively elucidated. Here, we report how PCNA SUMOylation and/or polyubiquitination affects recombination between direct repeats in S. cerevisiae. Our results show that these PCNA modifications primarily affect gene conversion, whereas their effect on the recombination-mediated deletion of intervening sequence is much less obvious. Siz1-dependent PCNA SUMOylation strongly limits Rad52/Rad51/Rad59-dependent gene conversion. A 5- to 10-fold increase in the frequency of such recombination events is observed in Siz1-defective strains, but this increase is fully suppressed when PCNA polyubiquitination is also compromised. PCNA polyubiquitination can stimulate gene conversion in both PCNA SUMOylation-proficient and SUMOylation-deficient strains. On the other hand, in PCNA polyubiquitination-deficient strains, the lack of PCNA SUMOylation does not affect GC levels. Therefore, we postulate that the antirecombinogenic activity of Siz1 mainly concerns recombination induced by PCNA polyubiquitination. In the absence of PCNA SUMOylation, the frequency of PCNA polyubiquitination-mediated gene conversion is not only increased, but it is also channeled into the Rad59-dependent pathway. Additionally, we show a weak inhibitory effect of Rad5 on Rad52/Rad59-directed single-strand annealing.

576. DNA Ends Matter: The Impact of Using CRISPR/Cas9 Variants on DNA Repair Pathway Choices and Editing Profiles at the HBB Locus

Sickle Cell Anemia is an inherited recessive disorder caused by a single point mutation in the human beta globin (HBB) gene resulting in an abnormal type of hemoglobin. Here we report targeting the HBB locus using CRISPR/Cas9 technology for correction of Sickle Cell Disease, which affects nearly 1 million people worldwide. Cas9 and its variants can be used to introduce a variety of breaks including blunt double stranded break (DSB), single nicks, or dual nicks leaving either a 3’ or 5’ overhang. The type of cut and donor used can play a role in triggering different repair pathways, thus, resulting in various editing profiles. Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes including indel mutations resulting from Non Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) using the donor as a template, and, finally, Gene Conversion (a kind of HDR event) using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene that corrects the sickle cell point mutation. We observed that the Cas9 nickase/gRNA pair leaving a 5’ overhang displayed a significantly higher frequency of gene conversion and gene correction than other Cas9-induced DNA end structures. We also provide evidence that overexpression or down-regulation of critical factors in the repair pathways influence the repair pathway balance.In summary, we demonstrate that the frequency of various repair outcomes under different conditions offers insight into the mechanisms of repair of Cas9-induced DNA cleavage. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.

Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant.

Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

Systematic characterization and comparative analysis of the rabbit immunoglobulin repertoire.

Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice.

Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination

Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.

Targeting DNA With Fingers and TALENs.

Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1,2 Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3,4,5 ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6 However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7,8,9,10 Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy.