Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability.

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.

A simple and novel DNA combing methodology for Fiber-FISH and optical mapping.

Single molecule analysis can help us study genomics efficiently. It involves studying single DNA molecules for genomic studies. DNA combing is one of such techniques which allowed us to study single DNA molecules for multiple uses. DNA combing technology can be used to perform Fiber-FISH and optical mapping. Physical mapping of genomes can be studied by restriction digestion of combed DNA on glass slides. Restriction fragments can be arranged into optical maps by gathering fluorescent intensity data by CCD camera and image analysis by softwares. Physical mapping and DNA segment rearrangements can be studied by Fiber-FISH which involves application of probes on genomic DNA combed over glass slides. We developed a novel methodology involving combing solution optimization, denatured combed DNA and performed restriction digestion of combed DNA. Thus we provided an efficient and robust combing platform for its application in Fiber-FISH and optical mapping.

Enhanced post wash retention of combed DNA molecules by varying multiple combing parameters.

Recent advances in genomics have created a need for efficient techniques for deciphering information hidden in various genomes. Single molecule analysis is one such technique to understand molecular processes at single molecule level. Fiber- FISH performed with the help of DNA combing can help us in understanding genetic rearrangements and changes in genome at single DNA molecule level. For performing Fiber-FISH we need high retention of combed DNA molecules post wash as Fiber-FISH requires profuse washing. We optimized combing process involving combing solution, method of DNA mounting on glass slides and coating of glass slides to enhance post-wash retention of DNA molecules. It was found that average number of DNA molecules observed post-wash per field of view was maximum with our optimized combing solution. APTES coated glass slides showed lesser retention than PEI surface but fluorescent intensity was higher in case of APTES coated surface. Capillary method used to mount DNA on glass slides also showed lesser retention but straight DNA molecules were observed as compared to force flow method.

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report.

BACKGROUND: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation. CASE PRESENTATION: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected. CONCLUSION: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.

Design of a microchannel-nanochannel-microchannel array based nanoelectroporation system for precise gene transfection.

A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 µm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.

Simultaneous Visualization of R-Loops/RNA:DNA Hybrids and Replication Forks in a DNA Combing Assay.

R-loops, structures that play a crucial role in various biological processes, are integral to gene expression, the maintenance of genome stability, and the formation of epigenomic signatures. When these R-loops are deregulated, they can contribute to the development of serious health conditions, including cancer and neurodegenerative diseases. The detection of R-loops is a complex process that involves several approaches. These include S9.6 antibody- or RNAse H-based immunoprecipitation, non-denaturing bisulfite footprinting, gel electrophoresis, and electron microscopy. Each of these methods offers unique insights into the nature and behavior of R-loops. In our study, we introduce a novel protocol that has been developed based on a single-molecule DNA combing assay. This innovative approach allows for the direct and simultaneous visualization of RNA:DNA hybrids and replication forks, providing a more comprehensive understanding of these structures. Our findings confirm the transcriptional origin of the hybrids, adding to the body of knowledge about their formation. Furthermore, we demonstrate that these hybrids have an inhibitory effect on the progression of replication forks, highlighting their potential impact on DNA replication and cellular function.

In Vivo Analysis of mtDNA Replication at the Single Molecule Level and with High Resolution.

Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.

Single-molecule analysis of mtDNA replication with high resolution.

DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.

Micromachining of Polymeric Microfluidic Micro/Nanoelectroporation Device.

Micro/nanochannel electroporation can deliver gene/drug into single cell with precise dosage control and much higher cell viability compared to traditional bulk electroporation. However, single cell micro/nanochannel electroporation has the problems of low efficiency and complicated operation. By integrating microfluidic with micro/nanochannel electroporation, a large number of cells can be processed within a short time. In this chapter, we provide a detailed protocol of fabrication microfluidic nanochannel electroporation devices. The fabrication of this microfluidic nanochannel electroporation device integrates soft lithography, DNA combing and imprinting, and micromilling. This device is appropriate for gene/drug delivery to a batch of cells. It has the advantages of both the single cell nanochannel electroporation and microfluidic based cell manipulation. The procedures of device fabrication, holder fabrication, cell trapping, and electroporation are included in this protocol.

Direct Visualization of DNA Replication at Telomeres Using DNA Fiber Combing Combined with Telomere FISH.

The ability to analyze individual DNA fibers undergoing active DNA synthesis has emerged as a powerful technique in the field of DNA replication. Much of the initial analysis has focused on replication throughout the genome. However, more recent advancements in this technique have allowed for the visualization of replication patterns at distinct loci or regions within the genome. This type of locus-specific resolution will greatly enhance our understanding of the dynamics of DNA replication in regions that provide a challenge to the replication machinery. Here, we describe a protocol that will allow for the visualization of DNA replication through one of the most structurally complex regions in the human genome, the telomeric DNA.

Genome wide decrease of DNA replication eye density at the midblastula transition of Xenopus laevis.

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.

Single Molecule Analysis of Resection Tracks.

Homologous recombination is initiated by the so-called DNA end resection, the 5'-3' nucleolytic degradation of a single strand of the DNA at each side of the break. The presence of resected DNA is an obligatory step for homologous recombination. Moreover, the amount of resected DNA modulates the prevalence of different recombination pathways. In different model organisms, there are several published ways to visualize and measure with more or less detail the amount of DNA resected. In human cells, however, technical constraints hampered the study of resection at high resolution. Some information might be gathered from the study of endonuclease-created DSBs, in which the resection of breaks at known sites can be followed by PCR or ChIP. In this chapter, we describe in detail a novel assay to study DNA end resection in breaks located on unknown positions. Here, we use ionizing radiation to induce double-strand breaks, but the same approach can be used to monitor resection induced by different DNA damaging agents. By modifying the DNA-combing technique, used for high-resolution replication analyses, we can measure resection progression at the level of individual DNA fibers. Thus, we named the method Single Molecule Analysis of Resection Tracks (SMART). We use human cells in culture as a model system, but in principle the same approach would be feasible to any model organism adjusting accordingly the DNA isolation part of the protocol.

Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing.

DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA-protein interaction, and DNA methylation along genomic single DNA molecules at 1 kb resolution. However, DNA combing has been restricted to short 200-500 kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.

Replication dynamics: biases and robustness of DNA fiber analysis.

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.