Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.     

Modulation of cell signaling pathways by kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, has been associated with the development of Kaposi's sarcoma, pleural effusion lymphoma, and multicentric Castleman's disease. KSHV is a double-stranded DNA virus that has been classified as a gammaherpesvirus. The viral genome is approx, 160 kb long and encodes for several genes that are involved in cell signaling pathways. These include genes that are unique to the virus as well as viral homologues of cellular genes. The latter are likely to have been usurped from the host genome and include both virokines and viral receptor proteins. This article reviews how these KSHV proteins modulate cellular signal transduction pathways.     

Primary products of break-induced recombination by Escherichia coli RecE pathway.

Alternative models for break-induced recombination predict different distributions of primary products. The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products. The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences. Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombination pathway of Escherichia coli. Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination products was determined. Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model. However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3' at the break. A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5' at the break (5' heteroduplexes) was observed at a larger stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5' heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks.     

Host cell DNA repair pathways in adeno-associated viral genome processing

Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PK(CS), but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.     

Stimulation of intrachromosomal homologous recombination in mammalian cells by an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose) polymerase (E.C. 2.4.2.30), on intrachromosomal homologous recombination in mouse Ltk- cells. We used a cell line that contained in its genome two defective Herpes thymidine kinase (tk) genes as closely linked direct repeats. Intrachromosomal homologous recombination events were monitored by selecting for tk-positive segregants that arose during propagation of the cells and recombination rates were determined by fluctuation analysis. We found that growth of cells in the continuous presence of 2mM 3-MB increased intrachromosomal recombination between 3 and 4-fold. Growth of cells in the presence of 2mM m-anisic acid, a non-inhibitory analog of 3-MB, had no effect on intrachromosomal recombination rates. Additionally, we found that 3-MB increased both gene conversions and crossovers to similar extents, adding to the evidence that these two types of intrachromosomal rearrangements share a common pathway. These findings contrast with our previous studies [Waldman, B.C. and Waldman, A.S. (1990) Nucleic Acids Res., 18, 5981-5988] in which we determined that 3-MB inhibits illegitimate recombination and has no effect on extrachromosomal homologous recombination in mouse Ltk- cells. An hypothesis is offered that explains the influence of 3-MB on different recombination pathways in mammalian cells in terms of the role that poly(ADP-ribosylation) plays in DNA break-repair.     

Is phage DNA 'injected' into cells--biologists and physicists can agree

The double-stranded DNA inside bacteriophages is packaged at a density of approximately 500 mg/ml and exerts an osmotic pressure of tens of atmospheres. This pressure is commonly assumed to cause genome ejection during infection. Indeed, by the addition of their natural receptors, some phages can be induced in vitro to completely expel their genome from the virion. However, the osmotic pressure of the bacterial cytoplasm exerts an opposing force, making it impossible for the pressure of packaged DNA to cause complete genome ejection in vivo. Various processes for complete genome ejection are discussed, but we focus on a novel proposal suggesting that the osmotic gradient between the extracellular environment and the cytoplasm results in fluid flow through the phage virion at the initiation of infection. The phage genome is thereby sucked into the cell by hydrodynamic drag.     

The cellular mismatch repair system is able to repair mismatches within MLV retroviral double-stranded DNA at a low frequency

Eukaryotic cells possess several distinct mismatch repair pathways. A mismatch can be introduced in retroviral double-stranded DNA by a pre-existing mutation within the primer binding site (PBS) of the viral RNA genome. In order to evaluate mismatch repair of retroviral double-stranded DNA, Moloney leukemia virus (MLV)-based vectors with a mutation in their PBS were used to infect mismatch repair-competent as well as mismatch repair-deficient cell lines. If the target cells were capable of repairing the mismatch before an infected cell divided, the mismatch within the PBS could be repaired to the wild-type or mutant PBS. If the target cells were unable to repair the mismatch, half the cells in the colony should contain the mutant PBS while the other half should contain the wild-type PBS. To evaluate these predictions, individual colonies were isolated and analyzed by PCR. Almost all mismatch-deficient cell colonies analyzed (cell lines HCT 116 and PMS2–/–) contained both the wild-type and mutated PBS, therefore, mismatches within retroviral double-strand DNA could not be repaired by the mismatch-deficient cells. In contrast, mismatches in ~25% of the mismatch repair-competent cell clones analyzed (cell lines HeLa and PMS2+/+) were repaired, while 75% were not. Therefore, the cellular mismatch repair system is able to repair mismatches within viral double-stranded DNA, but at a low frequency.     

Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or lowrisk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA. Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.     

A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Defective Interference in the Killer System of Saccharomyces cerevisiae

The K₁ killer virus (or plasmid) of Saccharomyces cerevisiae is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics 74:571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics 84:27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus (MAK genes) were needed by a suppressive mutant. No effect of ski mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.     

Bacterial Nonhomologous End Joining Requires Teamwork

All living organisms must repair DNA double-stranded breaks (DSBs) in order to survive. Many bacteria rely on nonhomologous end joining (NHEJ) when only a single copy of the genome is available and maintain NHEJ pathways with a minimum of two proteins. In this issue, Bhattarai and colleagues identify additional factors that can work together to aid in survival of stationary-phase cells with chromosomal breaks.     

Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.     

Human AP endonuclease inefficiently removes abasic sites within G4 structures compared to duplex DNA

Excision repair processes are essential to maintain genome stability. A decrease in efficiency and fidelity of these pathways at regions of the genome that can assume non-canonical DNA structures has been proposed as a possible mechanism to explain the increased mutagenesis and consequent diseased state frequently associated with these sites. Here we describe the development of a FRET-based approach to monitor the presence of G quadruplex (G4) DNA, a non-canonical DNA structure formed in runs of guanines, in damage-containing single-stranded and double-stranded DNA. Using this approach, we directly show for the first time that the presence within the G4 structure of an abasic site, the most common lesion spontaneously generated during cellular metabolism, decreases the efficiency of human AP endonuclease activity and that this effect is mostly the result of a decreased enzymatic activity and not of decreased binding of the enzyme to the damaged site. This approach can be generally applied to dissecting the biochemistry of DNA repair at non-canonical DNA structures.     

Global detection of DNA repair outcomes induced by CRISPR–Cas9

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.     

自身互补型腺相关病毒载体发展趋势

重组腺相关病毒(Recombinant adeno-associated virus,rAAV)可以作为基因运载工具将目的基因运送入靶器官并对多种疾病发挥治疗作用。以rAAV为载体进行基因治疗的关键是病毒基因组由单链变为双链,否则不能适时、有效表达目的基因。自身互补型rAAV(scrAAV)载体基因组本身以双链形式存在,与常规的单链rAAV(ssrAAV)载体相比,无论在表达时间还是表达强度上都有十分明显改善,可显著降低在疾病治疗过程中由于载体本身所诱发的免疫反应。目前,scrAAV已经在肝脏疾病、中枢神经系统疾病、眼部疾病、干细胞修饰以及RNA干扰、核酶技术等领域得到应用。以下在介绍scrAAV载体构建、表达、定位的基础上,以血友病B为主要对象,阐述scrAAV的应用潜力及发展趋势。