Surrogate reporters for enrichment of cells with nuclease-induced mutations

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Trashing the genome: the role of nucleases during apoptosis

Two classes of nucleases degrade the cellular DNA during apoptosis. Cell-autonomous nucleases cleave DNA within the dying cell. They are not essential for apoptotic cell death or the life of the organism, but they might affect the efficiency of the process. By contrast, waste-management nucleases are essential for the life of the organism. In post-engulfment DNA degradation, the DNA of apoptotic cells is destroyed in lysosomes of the cells that have phagocytosed the corpses. Waste-management nucleases also destroy DNA that is released into the extracellular compartment. Here, we describe the complex group of nucleases that are involved in DNA destruction during apoptotic cell death.     

DNA Segments Sensitive to Single-Strand-Specific Nucleases Are Present in Chromatin of Mitotic Cells

It was observed before that DNAin situin chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNAin situin mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells’ exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.     

哺乳动物基因组靶向修饰阳性细胞富集的报告载体系统研究进展

基因组靶向修饰技术对基因功能研究、基因治疗以及转基因育种研究都具有重要的意义和价值。近年来发展起来的人工核酸酶如ZFNs、TALENs和CRISPR/Cas9等的应用大大提高了基因组靶向修饰的效率。但是由于核酸酶表达载体转染效率、核酸酶表达效率及活性以及基因组被打靶后的修复效率等因素在一定程度上制约着基因组靶向修饰阳性细胞的获得。因此富集和筛选基因组靶向修饰阳性细胞是一个亟待解决的问题。报告载体系统可以间接地反映核酸酶的工作效率并有效富集核酸酶修饰的阳性细胞,进而提高基因组靶向修饰阳性细胞的富集和筛选效率。本文主要针对由非同源末端连接(Non-homologous end joining,NHEJ)和单链退火(Single-strand annealing,SSA)两种修复机制分别介导的报告载体系统的原理和应用进行了详细的介绍,以期为以后的相关研究提供借鉴和参考。     

Properties of enzymes that recognize modified structures in DNA

The properties of the major classes of DNA repair enzymes, such as DNA glycosylases, AP-endonucleases, incision nucleases, and alkyl transferases, are reviewed. With the exceptions of the incision nucleases, the properties of the enzymes are quite similar in prokaryotic and eukaryotic cells. The incision nucleases probably do not recognize the modified base residues as such, but rather helical distortion brought about by the modifying agents. The other classes of enzymes are more or less specific for certain modified structures.     

Cuts can kill: the roles of apoptotic nucleases in cell death and animal development

Chromosome fragmentation is one of the major biochemical hallmarks of apoptosis. However, until recently, its roles in apoptosis and mechanisms of action remained elusive. Recent biochemical and genetic studies have shown that chromosome fragmentation is a complex biochemical process that involves a plethora of conserved nucleases with distinct nuclease activities and substrate specificities. These apoptotic nucleases act cooperatively among themselves and with other nonnuclease cofactors to promote stepwise chromosome fragmentation and DNA degradation. Importantly, in addition to its direct contribution to the dismantling of the dying cell, apoptotic DNA degradation can facilitate cell killing and other apoptotic events such as clearance of apoptotic cells. Furthermore, some apoptotic nucleases apparently affect other aspects of animal development, including immune responses. The identification of new apoptotic nucleases and analysis of their functions in apoptosis and animal development should pave the way for future studies to uncover new functions for apoptotic nucleases and shed light on the hidden links between apoptotic DNA degradation and human diseases.     

Nuclease content of group H Streptococcus strain Challis cell surface extracts inactivating transforming deoxyribonucleic acid.

Besides the competence factor (cpf), the activity of nuclease present in cell surface extracts (iF) is most likely necessary for the occurrence of competence in transformation of Challis strain and other group H streptococci. Very small amounts of iF activity were available. For this reason there were no data on nucleases occurring in iF preparations. In our previous studies, three deoxyribonucleases (endonucleases) were isolated and partly purified from stationary Challis strain cells. In the present work, due to the application of stationary-phase cell endonucleases, gel electrophoresis, and the immunological method, we found that two of these nucleases occur in Challis strain iF preparations. These two nucleases are present in larger amounts at the cell surface only in cells originating from the early-logarithmic-phase culture. Only in this phase does competence occur in the Challis strain. Antibodies against the three endonucleases of the Challis strain do not block the occurrence of competence. We suggest that an increase in the permeability of the cell membrane for intracellular nucleases independent of cpf activity is a stage in the maturation of competence.     

Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells

Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.     

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.     

Mechanism of the radioprotective effect of cysteamine

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.