FANCJ helicase controls the balance between short- and long-tract gene conversions between sister chromatids.

The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.

Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.

Mitochondrial diseases are commonly caused by mutated mitochondrial DNA (mtDNA), which in most cases coexists with wild-type mtDNA, resulting in mtDNA heteroplasmy. We have engineered transcription activator-like effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. Mitochondria-targeted TALEN (mitoTALEN) expression led to permanent reductions in deletion or point-mutant mtDNA in patient-derived cells, raising the possibility that these mitochondrial nucleases can be therapeutic for some mitochondrial diseases.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR ß- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining.

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.

Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells.

Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.

Fluorescent tagged analysis of neural gene function using mosaics in zebrafish and Xenopus laevis.

An important question in the neurosciences is the role of specific gene expression in the control of neural morphology and connectivity. To address this question, methods are needed for expression of exogenous genes in a subset of neurons. This limited and mosaic expression allows the assessment of gene expression in a cell autonomous fashion without environmental contributions from neighboring expressing cells. These methods must also label neurons so that detailed morphology and neural connections can be evaluated. The labeling method should label only a subset of neurons so that neuronal morphology can be viewed upon a non-stained background, in a Golgi staining fashion. Here, we report methods using plasmids called pTAGUM (tagged analysis of genes using mosaics) that accomplish these goals. These methods should prove useful for the analysis of neural gene function in two important model organisms, the zebrafish and Xenopus laevis.

Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends.

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.

Enhanced stimulation of chromosomal translocations and sister chromatid exchanges by either HO-induced double-strand breaks or ionizing radiation in Saccharomyces cerevisiae yku70 mutants.

DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MATa and MATalpha inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs is enhanced in haploids and diploids expressing both MATa and MATalpha genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1::his3-Delta3'::HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent.

Association between vitamin D receptor FokI. Polymorphism and serum parathyroid hormone level in patients with chronic renal failure.

We investigated the relationship between vitamin D receptor (VDR) start codon polymorphism and serum levels of PTH, calcidiol, and calcium in 64 Spanish patients with chronic renal failure (CRF). An exon 2 fragment of the VDR gene was amplified by PCR, and cleaved with the restriction enzyme FokI. The alleles were identified according to the digestion pattern obtained as F (absence of restriction site) and f (presence of restriction site). Genotype frequencies in the patient population were 54.7% FF, 28.1% Ff and 17.2% ff, vs 46.7% FF, 43.3% Ff and 10% ff in a healthy control population. The difference between the two populations was statistically significant (p<0.01). Within the patient population, mean serum PTH level in the FF group was significantly higher (159.77+/-25.69 pg/ml) than in both the Ff and ff groups (106.67+/-19.07 and 77.55+/-15.85 pg/ml, respectively; p<0.05). However there were no significant differences in serum levels of calcidiol or calcium among genotypes. These results suggest that FokI polymorphisms of the VDR gene may determine parathyroid response in CRF patients.

Improving gene replacement by intracellular formation of linear homologous DNA.

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.

[Influence of combined microinjection of gene engineering construct and endonuclease on preimplantation development of mouse embryos in vitro]. / Vliianie sovmestnoi mikroin''ektsii genno-inzhenernoi konstruktsii i éndonukleazy na doimplantatsionnoe razvitie émbrionov myshi in vitro.

We studied the influence of combined microinjection of a gene engineering construct and site-specific endonuclease Sal in the pronucleus on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of survival of the embryos was estimated according to their capacity to develop until the blastocyst stage and hatch from zona pellucida. The results obtained suggest that the microinjection of exogenous DNA jointly with endonuclease SalI at concentrations from 0.1 to 0.01 U/microl decreased reliably the rate of survival, as compared to the control (p < 0.05 and p < 0.01, respectively). However, a decrease of endonuclease SalI concentration in the injection mixture to 0.01 U/microl enhanced the capacity of mouse embryos to develop until the blastocyst stage and hatch from zona pellucida, as compared to the embryos microinjected with exogenous DNA and endonuclease SalI at a higher concentration.

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis.

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.

[Application of the Bgl II-Bln I dosage test to gene diagnosis of facioscapulohumeral muscular dystrophy 1A gene].

OBJECTIVE: To increase the sensitivity and specificity of conventional gene diagnosis of facioscapulohumeral muscular dystrophy 1A(FSHD1A) by analyzing the distribution of translocation between chromosomes 4q35 and 10q26 in suspected FSHD cases. METHODS: The Bgl II- Bln I dosage test was performed to detect translocation between chromosomes 4q35 and 10q26 in 7 cases of presymptomatic FSHD patients showing positive result in gene diagnosis and 5 cases of sporadic FSHD patients showing negative result in gene diagnosis. DNA samples were digested with Bgl II and Bln I, followed by agrose gel electrophoresis. Probe p13E-11 was labeled with alpha-(32) P dCTP, followed by Southern hybridization. Then the ratio between the chromosomes 4 and 10 derived signal intensities was judged and hence was made known whether there was interchromosomal translocation between chromosomes 4 and 10. RESULTS: The Bgl II-Bln I dosage test revealed a translocation from chromosome 4q35 to 10q26 in one presymptomatic FSHD patient, thus indicating the result of gene diagnosis for her might be false positive. There was one translocation from chromosome 10q26 to 4q35 detected in one sporadic FSHD patient, indicating the result of gene diagnosis for her might be false negative. There were no translocations between chromosomes 4 and 10 in the other 10 cases. CONCLUSION: The Bgl II-Bln I dosage test can detect the translocation between chromosomes 4q35 and 10q26. It can improve the accuracy of the conventional method for gene diagnosis of FSHD1A.

Progress towards understanding the nature of chromatid breakage.

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome.

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

A novel L94Q mutation in the CDKN2A gene in a melanoma kindred.

About 10% of melanoma cases have clinical factors indicative of hereditary cancer. CDKN2A is a major melanoma susceptibility gene in familial malignant melanoma. In this study a novel L94Q missense mutation of the CDKN2A gene is described in a melanoma kindred with two affected second-degree family members. To detect the mutation, polymerase chain reaction (PCR) amplification methods and direct sequencing were used. The presence of the mutation was confirmed by restriction fragment length polymorphism analysis after digestion of the PCR amplicons with the restriction endonuclease BspMI. The penetrance of the novel mutation was shown to be incomplete. Functional importance of the mutation was assumed from the protein p16 structure.

High throughput creation of recombinant adenovirus vectors by direct cloning, green-white selection and I-Sce I-mediated rescue of circular adenovirus plasmids in 293 cells.

Ability of replication-defective adenovirus vectors to achieve efficient gene transfer in most of the mammalian cell types makes them useful vehicles for many gene transfer applications, including their use in assessing gene function. High throughput creation of recombinant adenovirus becomes a critical path to the expanding utility of adenovirus vector technology. Here, we report a process in which recombinant adenovirus vectors are isolated as single molecular clones through a convenient direct cloning and green-white selection procedure, and directly transfected into 293 cells where virus is rescued through an enzymatic reaction mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular clones was at least 10-fold more efficient than that using linearized clones for transfection. This method will facilitate a high throughput creation of vectors as required for screening gene function.

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast.

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.

Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone.

The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed.

Novel fluorescent ligase detection reaction and flow cytometric analysis of SYT-SSX fusions in synovial sarcoma.

Synovial sarcomas (SS) are characterized by the t(X;18)(p11;q11) translocation and its resultant fusion gene, SYT-SSX. Two homologues of the SSX gene (ie, SSX1 and SSX2) are involved in the vast majority of SS and the SYT-SSX1 type of fusion has been associated with inferior clinical outcome. Thus, detection of the presence and type of SYT-SSX fusion is critical for diagnosis and prognosis in SS. Identification of SYT-SSX fusion type is typically accomplished by reverse-transcription polymerase chain reaction (RT-PCR) followed by a post-PCR analytic method. As mRNA nucleotide sequences of the SSX1 and SSX2 segments involved in the SYT-SSX fusion are nearly identical, post-PCR methods must be highly discriminatory. We describe a novel method to identify and differentiate these two chimeric transcripts using RT-PCR followed by fluorescent thermostable ligase detection reaction (f-LDR), microparticle bead capture and flow cytometric detection. Evaluation of this unique approach in 11 cases of SS without prior knowledge of SYT-SSX status, six cases of control sarcomas (CS) and three hematopoietic cell lines, revealed that the f-LDR technique was rapid, unambiguous, and highly specific. The f-LDR results were compared to XmnI enzyme digestion patterns and sequencing of PCR products, revealing a 100% concordance for all cases of SS with regards to SYT-SSX transcript type. In addition, there was a strong association of transcript type detected by f-LDR and morphological subclassification of SS, as previously reported. We conclude that this f-LDR method with flow-based detection is a robust approach to post-PCR detection of specific nucleotide sequences in SS and may be more broadly applicable in molecular oncology.