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Biomedical subjects

E B Kmiec

Publications and source records attributed to E B Kmiec.

At least 19 recordsLinked to original sources
PubMed2005

Gene therapy progress and prospects: targeted gene repair.

The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models.

DNA Repair↗
PubMed2004

Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting.

A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.

Base Sequence↗
PubMed2001

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides.

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.

Base Sequence↗
PubMed2001

Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors.

Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.

Animals↗
PubMed2001

The potential of nucleic acid repair in functional genomics.

Chimeric RNA/DNA oligonucleotides have been used successfully to correct point and frameshift mutations in cells as well as in animal and plant models. This approach is one of several nucleic acid repair technologies that will help elucidate the function of newly discovered genes. Understanding the mechanisms by which these different technologies direct gene alteration is essential for progress in their application to functional genomics.

DNA↗
PubMed2001

In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae.

Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has proven successful in eukaryotic cells including animal and plant models. In many cases, however, there has been a disparity in the levels of gene correction or frequency. While the delivery of these chimera into the nucleus and the long-term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the key to improving or stabilizing frequency. To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experiments in cell-free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors. RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppressive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, containing a marker gene, were converted to wild type, evidenced by the expression of green fluorescence in converted cells. Because the repaired fusion gene contains a corrected neomycin sequence, cells were subsequently placed under G418 selection and conversion confirmed at the genetic level. Taken together, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for evaluating true frequencies of correction. Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling measurements of conversion in real time.

Base Sequence↗
PubMed2001

Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides.

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell-free extracts are competent to support oligonucleotide-directed genetic repair. Using this system, we are studying Arabidopsis DNA-repair mutants and the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA-repair pathways.

Arabidopsis↗
PubMed2000

Evidence for a four-strand exchange catalyzed by the RecA protein.

Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.

Adenosine Triphosphate↗
PubMed2000

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts.

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Animals↗
PubMed2000

A plausible mechanism for gene correction by chimeric oligonucleotides.

Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.

Cell-Free System↗
PubMed2000

HsRec2/Rad51L1, a protein influencing cell cycle progression, has protein kinase activity.

Human Rec2/Rad51L1 is a member of the Rad51 family of proteins. Although recombinase activity, typical of this family, could not be established, its overexpression in mammalian cells has been shown to cause a delay in G1. Moreover, since hsRec2/Rad51L1 has been found to be induced by both ionizing and UV irradiation, it is likely that hsRec2/Rad51L1 is elevated following any DNA damage and causes a G1 delay to allow time for DNA repair to occur. Limited homology with catalytic domains X and XI of protein kinase A suggested that kemptide, an artificial substrate containing one phosphorylatable residue, a serine, might serve as a substrate for hsRec2/Rad51L1. Here, we report that hsRec2/Rad51L1 can phosphorylate kemptide, as well as myelin basic protein, p53, cyclin E, and cdk2, but not a peptide substrate containing tyrosine only. The finding that hsRec2/Rad51L1 exhibits protein kinase activity is a first step toward identifying a mechanism whereby this protein affects the cell cycle.

CDC2-CDC28 Kinases↗
PubMed2000

Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides.

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.

Base Sequence↗
PubMed1999

Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract.

Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo. Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, we developed a system to study the mechanisms of chimera-directed gene conversion. Using a mammalian cell-free extract and a genetic readout in Escherichia coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively. The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.

Animals↗
PubMed1999

Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background.

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.

Animals↗
PubMed1998

Analysis of the human RAD51L1 promoter region and its activation by UV light.

The human REC2/RAD51B gene (HGMW-approved symbol RAD51L1) encodes a 350-amino-acid protein with regional homologies to members of the RAD52 epistasis group. It is induced by DNA-damaging agents, and the overexpression of this gene product causes G1/S cell cycle arrest. In this report, the promoter region, containing the UV-responsive element, is revealed. Deletion analyses of a 1699-base fragment at the 5' end of the human REC2/RAD51B cDNA identified a 116-base sequence that appears to be responsible for radiation induction. This fragment contains many DNA sequences that have been identified in the promoter regions of other radiation-inducible genes in yeast and humans. Within this region are "consensus" binding sites for both the AP2 and the p53 proteins that may act to regulate the expression of the human REC2/RAD51B gene. Five putative transcripts have been identified from regions 5' of the promoter element that splice near the ATG translation start site. None of the transcripts contain the UV-inducible element nor the consensus transcription factor binding sites.

Base Sequence↗