Ends Of Linear DNA Research Articles

SbcB facilitates natural transformation in Vibrio cholerae in an exonuclease-independent manner.

Horizontal gene transfer by natural transformation contributes to the spread of antibiotic resistance and virulence factors in bacterial species. Here, we study how one protein, SbcB, helps facilitate this process in the facultative bacterial pathogen Vibrio cholerae . SbcB is a well-known for its exonuclease activity ( i.e ., the ability to degrade the ends of linear DNA). Through this study we uncover that while SbcB is important for natural transformation, it does not facilitate this process using its exonuclease activity. Thus, this work helps further our understanding of the molecular events required for this conserved evolutionary process, and uncovers a function for SbcB beyond its canonical exonuclease activity.

High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected—between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration.

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

DNA in nanochannels: theory and applications.

Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

Proximal-end bias from in-vitro reconstituted nucleosomes and the result on downstream data analysis.

The most basic level of eukaryotic gene regulation is the presence or absence of nucleosomes on DNA regulatory elements. In an effort to elucidate in vivo nucleosome patterns, in vitro studies are frequently used. In vitro, short DNA fragments are more favorable for nucleosome formation, increasing the likelihood of nucleosome occupancy. This may in part result from the fact that nucleosomes prefer to form on the terminal ends of linear DNA. This phenomenon has the potential to bias in vitro reconstituted nucleosomes and skew results. If the ends of DNA fragments are known, the reads falling close to the ends are typically discarded. In this study we confirm the phenomenon of end bias of in vitro nucleosomes. We describe a method in which nearly identical libraries, with different known ends, are used to recover nucleosomes which form towards the terminal ends of fragmented DNA. Finally, we illustrate that although nucleosomes prefer to form on DNA ends, it does not appear to skew results or the interpretation thereof.

Telomeric and Sub-Telomeric Structure and Implications in Fungal Opportunistic Pathogens.

Telomeres are long non-coding regions found at the ends of eukaryotic linear chromosomes. Although they have traditionally been associated with the protection of linear DNA ends to avoid gene losses during each round of DNA replication, recent studies have demonstrated that the role of these sequences and their adjacent regions go beyond just protecting chromosomal ends. Regions nearby to telomeric sequences have now been identified as having increased variability in the form of duplications and rearrangements that result in new functional abilities and biodiversity. Furthermore, unique fungal telomeric and chromatin structures have now extended clinical capabilities and understanding of pathogenicity levels. In this review, telomere structure, as well as functional implications, will be examined in opportunistic fungal pathogens, including Aspergillus fumigatus, Candida albicans, Candida glabrata, and Pneumocystis jirovecii.

Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion

We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets.

The gp44 Ejection Protein of Staphylococcus aureus Bacteriophage 80α Binds to the Ends of the Genome and Protects It from Degradation.

Bacteriophage 80α is a representative of a class of temperate phages that infect Staphylococcus aureus and other Gram-positive bacteria. Many of these phages carry genes encoding toxins and other virulence factors. This phage, 80α, is also involved in high-frequency mobilization of S. aureus pathogenicity islands (SaPIs), mobile genetic elements that carry virulence factor genes. Bacteriophage 80α encodes a minor capsid protein, gp44, between the genes for the portal protein and major capsid protein. Gp44 is essential for a productive infection by 80α but not for transduction of SaPIs or plasmids. We previously demonstrated that gp44 is an ejection protein that acts to promote progression to the lytic cycle upon infection and suggested that the protein might act as an anti-repressor of CI in the lytic–lysogenic switch. However, an 80α Δ44 mutant also exhibited a reduced rate of lysogeny. Here, we show that gp44 is a non-specific DNA binding protein with affinity for the blunt ends of linear DNA. Our data suggest a model in which gp44 promotes circularization of the genome after injection into the host cell, a key initial step both for lytic growth and for the establishment of lysogeny.

Rapid and Definitive Analysis of In Vitro DNA Methylation by Nano-electrospray Ionization Mass Spectrometry

CpG methylation of DNA is an epigenetic marker that is highly related to the regulation of transcription initiation. For analysis of CpG methylation in genomic DNA sequences, bisulfite-induced modification in combination with polymerase chain reaction (PCR) is usually utilized, but it cannot be straightforwardly applied to methylated short- and middle-sized DNAs, such as < 500 base pairs (bp), which are often utilized in structural biology studies. In the present study, we applied nano-electrospray ionization mass spectrometry (nano-ESI-MS) for the characterization of methylated DNA with < 400 bp prepared in vitro. First, double-stranded DNA oligomers were methylated with recombinant M.SssI DNA methylase, which has been reported to modify completely and exclusively CpG sites in the sequence. The fragments generated by the digestion with methylation-insensitive restriction nuclease were then analyzed to identify the methylation levels by nano-ESI-MS, without liquid chromatography (LC) separation. By methylation-insensitive nuclease digestion, we divided the DNA strands into several fragments, and nano-ESI-MS enabled the accurate analysis of methylation levels in the DNA fragments with a relatively small amount of DNA sample prepared under optimized conditions. Furthermore, it was revealed that M.SssI methylase hardly modifies the CpG sites closely positioned at the ends of linear DNA. The present method is similar to the strategy for post-translational modification analysis of proteins and is promising for the rapid and definitive characterization of methylated DNA that may be used in structural biology studies.

Telomeres Increasingly Develop Aberrant Structures in Aging Humans.

Telomeres progressively shorten with age, and it has been proposed that critically short and dysfunctional telomeres contribute to aging and aging-associated diseases in humans. For many years it was thought that telomere erosion was strictly a consequence of the "end replication problem," or the inability of replicative polymerases to completely duplicate linear DNA ends. It is becoming increasingly evident, however, that telomere shortening of cultured human cells is also caused because of other replication defects in telomeric repeats, those that cause fragile telomeres and other aberrant telomeric structures that can be detected on metaphase chromosomes. Whether these replication defects contribute to telomere erosion also in human tissues is currently unknown. By analyzing peripheral blood mononuclear cells from a total of 35 healthy subjects ranging in age from 23 to 101 years, we demonstrated that telomeres increasingly display aberrant structures with advancing donor age. Although the percentages of fragile telomeres increased only until adulthood, the percentages of chromosomes displaying sister telomere loss and sister telomere chromatid fusions increased consistently throughout the entire human life span. Our data, therefore, suggest that telomeric replication defects other than the end replication problem contribute to aging-associated telomere erosion in humans.

The Association of Early and Recent Psychosocial Life Stress With Leukocyte Telomere Length.

Chronic exposure to psychosocial stressors is related to worse somatic health. This association applies both to stressors early in life, such as childhood adversities, and more recent life stress, such as stressful life events. This study examined whether accelerated telomere shortening, as an indicator of cellular aging, might be an explanatory mechanism. We examined whether childhood adversities and recent stressful life events were associated with shorter telomeres in 2936 participants (mean [standard deviation] age = 41.8 [13.1] years, 66% women, 57% current depression) of the Netherlands Study of Depression and Anxiety. Telomeres are specialized nucleic acid-protein complexes at the ends of linear DNA that shorten with age; telomere length (TL) was measured with quantitative polymerase chain reaction. Childhood life events (β = .004, p = .805) and childhood trauma (β = -.023, p = .205) were not related to shorter TL. However, we found negative associations between recent stressful life events and TL. Persons had shorter telomeres if they reported more stressful life events in the past year (β = -.039, p = .028) and 1 to 5 years ago (β = -.042, p = .018, adjusted for sociodemographics). The relationship between stressful life events and TL became borderline significant when further adjusted for smoking status. No associations with TL were found when stressful life events occurred more than 6 years ago (p > .10). Results show that recent stressful life events are associated with shorter TL. This association is not observed for psychosocial stressors that occur earlier in life. Whether these results are indicative of physiological resiliency remains to be explored by future longitudinal research.

Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase

Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3′-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.

Dynamics of Plectonemic Supercoils along Stretched DNA by Brownian Dynamics

Interwound DNA structures (plectonemes) arise from processes such as replication, transcription and the nearly 10000-fold compaction of long DNA molecules into the cell nucleus. These supercoils can enhance or repress cellular processes such as replication, transcription, and recombination. In fact, an increased frequency of DNA-DNA juxtapositions within plectonemic supercoils facilitates DNA looping. Although plectonemes have been studied with recent single molecule techniques, little is known about their dynamic formation, diffusion, rearrangement, and dissolution. In fact, single molecule assays often are confounded by the relatively slow dynamics of a large magnetic or optical bead used to manipulate the DNA. To characterize plectoneme dynamics, we employ Brownian dynamics simulations of stretched supercoiled DNA. Here we consider a range of system parameters describing length, extension, and superhelical density. Our model incorporates viscous drag, thermal fluctuations, bending, torsion, extension, and electrostatics. Additionally, we employ periodic boundary conditions to prevent plectonemes from diffusing off the ends of linear DNA. Our simulations reveal that both the number and size of plectonemes vary with time. Interestingly, this precludes the characterization of plectoneme motion with a diffusion constant. To quantify plectoneme dynamics, we define τ as the median time for first juxtaposition of two sites on a DNA molecule separated by a prescribed distance. Here we show that τ depends on system parameters as well as separation distance and ranges from milliseconds to seconds. Our simulations shed light on processes requiring juxtaposition (e.g. DNA looping) as well as processes in which supercoils are transmitted along DNA (e.g. transcription).

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Catalytic Residues of the Telomere Resolvase ResT: A PATTERN SIMILAR TO, BUT DISTINCT FROM, TYROSINE RECOMBINASES AND TYPE IB TOPOISOMERASES

ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of co-valently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5'-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4-6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.

Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration.

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing endonuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and double-strand breaks were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the target cleavage site by imprecise nonhomologous end joining occurred at a frequency of approximately 10(-3) per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of double-strand break repair, we observed integration of viral DNA at the site of the break with a frequency of approximately 10(-4) per transfected cell. Integration depended on the production of viral double-stranded linear DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral linear DNA ends and the I-SceI-induced break, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes beyond that caused by viral DNA integration itself [corrected].

In vitro reconstitution of the end replication problem.

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.

The La antigen associates with the human telomerase ribonucleoprotein and influences telomere length in vivo.

La is an important component of ribonucleoprotein complexes and telomerase is a ribonucleoprotein that compensates for the shortening of the ends of linear DNA by adding telomeric repeats onto the ends of chromosomes by using an integral RNA as the template. We have identified a direct and specific interaction between La and the RNA component of human telomerase. Antibodies specific to La precipitate the human telomerase ribonucleoprotein complex derived from tumor cells, telomerase immortalized normal cells, and in vitro transformed cells. Overexpression of La in both experimentally immortalized human cells and prostate cancer cells results in gradual telomere shortening. Our results demonstrate that La can associate with telomerase and its expression level can influence telomere homeostasis in vivo.

Telomerase Can Inhibit the Recombination-based Pathway of Telomere Maintenance in Human Cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Kinetic Characterization of Linear Diffusion of the Restriction Endonuclease EcoRV on DNA

We have examined the kinetic parameters of linear diffusion of EcoRV on DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of linear diffusion, and the behavior of enzymes at the ends of linear DNA is explicitly treated. The analysis of the dependence of linear diffusion on the concentrations of NaCl and MgCl2 shows that linear diffusion is maximal at 50 mM NaCl under all concentrations of MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM, the highest concentration used in the test. Under these conditions, EcoRV scans 2 x 10(6) bp during one binding event with a velocity of about 1.7 x 10(6) bp s-1. The enzyme tends to overlook cleavage sites at 1 mM but not at 10 mM MgCl2. This result confirms the thermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and a specific binding mode of EcoRV to DNA. By comparing cleavage rates of linear DNA whose ends are free or blocked, we have shown that EcoRV has a very low probability to fall off at the ends of linear DNA. The enzyme rather is "reflected" and continues linear diffusion. EcoRV does not cleave oligonucleotides containing two EcoRV sites processively. Consequently, dissociation of the enzyme from the cleavage products is not preceded by a transfer to nonspecific DNA, and linear diffusion is not involved in product dissociation in EcoRV.