Mapping Protein-ssDNA Interactions at Single Nucleotide Resolution by Dual Nucleases-Assisted Next-Generation Sequencing.

The T4 bacteriophage helicase loader (gp59) is one of the main eight proteins that play an active role in the replisome. gp59 is a small protein (26 kDa) that exists as a monomer in solution and in the crystal. It binds preferentially to forked DNA and interacts directly with the T4 helicase (gp41), single-stranded DNA-binding protein (gp32), and polymerase (gp43). However, the stoichiometry and structure of the functional form are not very well understood. There is experimental evidence for a hexameric structure for the helicase (gp41) and the primase (gp61), inferring that the gp59 structure might also be hexameric. Various experimental approaches, including gel shift, fluorescence anisotropy, light scattering, and fluorescence correlation spectroscopy, have not provided a clearer understanding of the stoichiometry. In this study, we employed single-molecule photobleaching (smPB) experiments to elucidate the stoichiometry of gp59 on a forked DNA and to investigate its interaction with other proteins forming the primosome complex. smPB studies were performed with Alexa 555-labeled gp59 proteins and a forked DNA substrate. Co-localization experiments were performed using Cy5-labeled forked DNA and Alexa 555-labeled gp59 in the presence and absence of gp32 and gp41 proteins. A systematic study of smPB experiments and subsequent data analysis using a simple model indicated that gp59 on the forked DNA forms a hexamer. In addition, the presence of gp32 and gp41 proteins increases the stability of the gp59 complex, emphasizing their functional role in T4 DNA replication machinery.

A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Probing the Nucleic Acid Binding Properties of the Single-Stranded DNA Binding Protein of Bacteriophage T4 Replication Complex at Single Nucleotide Resolution

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

BioTechniquesVol. 54, No. 2 BioSpotlightOpen AccessBioSpotlightPatrick C.H. Lo & Kristie NyboPatrick C.H. LoSearch for more papers by this author & Kristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113985AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Breaking pointChromosomal rearrangements often disrupt specific genes, potentially leading to genetic disorders or syndromes. Defining the molecular nature of these syndromes often requires mapping the breakpoint locations, but traditional methods for mapping breakpoints such as fluorescence in situ hybridization (FISH) lack the resolution to precisely pinpoint a break at the nucleotide level. Multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing offer that single nucleotide resolution, but are laborious and time consuming. In the search for a more effective way to localize chromosomal breakpoints, Okoniewski et al. from the University of Zurich, Switzerland tested the capabilities of two next-generation sequencing platforms in identifying breakpoints, and present their conclusions in this issue of BioTechniques. The authors selected the Illumina HiSeq 2000 and PacBio RS platforms, which offer a significant number of short 100 bp reads and long 2 kb reads, respectively, and compared their abilities to characterize large deletions previously identified by MLPA and microarray analyses. The authors found that both platforms provided sufficient read depths to identify precise breakpoint locations at the nucleotide level with comparable sample preparation costs. The approaches differed in preparation time -- with the Illumina methodology requiring more time for sample preparation and sequencing -- and in the approach to breakpoint detection. With the Illumina system, breakpoints were seen as an increase in mismatches, in part because the short reads were able to map across the breakpoints since the mapping software allowed several mismatches. In contrast, PacBio data showed a clear drop of read depth in the deleted region. During this study, the authors found data analysis particularly challenging since few dedicated computational techniques were available for characterizing large deletions using next-generation sequencing. Therefore, Okoniewski et al. decided to create a new script to count matches at putative breakpoint sites. This script, along with the validating data for the Illumina HiSeq and PacBio platforms, comprise a new alternative for rapid breakpoint identification that requires fewer resources and less time than traditional Sanger sequencing.Experimental design of the miR-HTS method.See “Precise breakpoint localization of large genomic deletions using PacBio and Illumina next-generation sequencers”Functional miRNA screens using qPCRMicroRNAs (miRNAs) are involved in the regulation of developmental, physiological, and pathophysiological processes. Observing the differential expression of an miRNA during development or pathogenesis, however, does not prove a direct role for that miRNA in regulating the process since changes in abundance could be the consequence and not the cause. Functional miRNA screens can determine whether candidate miRNAs with altered expression levels are in fact regulating a specific biological process, and high-throughput functional assays are critical for screening large numbers of candidate miRNAs to identify clinically relevant targets. Such high-throughput screens typically take advantage of large libraries of miRNA mimics or lentiviral miRNA overexpression constructs that are individually arrayed in microplates, but this approach requires expensive robotics and is subject to the variations and errors inherent in large-scale, multiwell HTP screens. A simpler alternative is to use pooled lentiviral or retroviral miRNA libraries to infect a population of cells such that each infected cell only has a single virus overexpressing a unique miRNA or miRNA cluster. Specific miRNAs in the pool whose overexpression in cells then causes either over- or under-representation of a specific phenotype can be identified by quantitating unique miRNA expression cassette “barcodes”. Methods to quantify barcodes include next-generation sequencing, bead-based assays, and custom-made microarrays, but all of these approaches require expensive instrumention and complicated bioinformatic analyses. In this issue of BioTechniques, C. Civin and colleagues at the University of Maryland School of Medicine demonstrate how quantitative PCR can used as a simpler means to assay for specific miRNA barcodes in functional screens of pooled lentiviral miRNA libraries. As a proof of principle, the authors used their “miR-HTS” method to screen for miRNAs regulating cell growth in the IMR90 human lung fibroblast cell line using a pooled human lenti-miRNA library and custom qPCR assays. Fifty nine growth-inhibitory miRNAs were isolated, of which only four were previously identified as inhibiting growth in human lung cells. Nine of twelve miRNAs randomly selected from among the 55 novel candidate growth-inhibitory miRNAs were validated in follow-up experiments to inhibit IMR90 cell growth. This miR-HTS method is simpler, less expensive, and presents a more flexible approach for functional miRNA screening using pooled lenti- or retro-viral miRNA libraries.See “A simple high-throughput technology enables gain-of-function screening of human microRNAs”FiguresReferencesRelatedDetails Vol. 54, No. 2 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print February 2013 Information© 2013 Author(s)PDF download

The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

1 Targeted next generation sequencing (NGS) of the CFTR locus: Comparison of three technical approaches

Recent development of next generation sequencing (NGS) technologies has led to the identification of structural variants (SVs) of genomic DNA existing in the human population. Several SV detection methods utilizing NGS data have been proposed. However, there are several difficulties in analysis of NGS data, particularly with regard to handling reads from duplicated loci or low-complexity sequences of the human genome. In this paper, we propose SVEM, a novel statistical method to detect SVs with a single nucleotide resolution that can utilize multi-mapped reads on breakpoints. SVEM estimates the amount of reads on breakpoints as parameters and mapping states as latent variables using the expectation maximization algorithm. This framework enables us to handle ambiguous mapping of reads without discarding information for SV detection. SVEM is applied to simulation data and real data, and it achieves better performance than existing methods in terms of precision and recall.

DNA methylation in higher organisms has become an expanding field of study as it often involves the regulation of gene expression. Although Whole Genome Bisulfite Sequencing (WG-BS) based on next-generation sequencing (NGS) is the most versatile method, this is a costly technique that lacks in-depth analytic power. There are no conventional methods based on NGS that enable researchers to easily compare the level of DNA methylation from the practical number of samples handled in the laboratory. Although the targeted BS method based on Sanger sequencing is generally used in this case, it lacks in-depth analytic power. Therefore, we propose a new method that combines the high throughput analytic power of NGS and bioinformatics with the specificity and focus offered by PCR-amplification-based bisulfite sequencing methods. We use in silico size sieving of DNA-fragments and primer matchings instead of whole-fragment alignment in our bioinformatics analyses, and named our method SIMON (Simple Inference for Methylome based On NGS). The results of our targeted BS method based on NGS (SIMON method) show that small variations in DNA methylation patterns can be precisely and efficiently measured at a single nucleotide resolution. SIMON method combines pre-existing techniques to provide a cost-effective technique for in-depth studies that focus on pre-identified loci. It offers significant improvements with regard to workflow and the quality of the acquired DNA methylation information. Because of the high accuracy of the analysis, small variations of DNA methylation levels can be precisely determined even with large numbers of samples and loci.

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.

The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.