Single molecule analysis of DNA replication

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.     

Single molecule conformational analysis of DNA G-quadruplexes

Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.     

Single large DNA molecule analysis using fluorescence microscopy.

A large DNA analysis method which enable to obtain spatial information of positions of specific sequences along DNA molecule has been developed. Making use of the phenomenon that large DNA molecule is elongated stably under alternative current field in a concentrated linear polymer solution, direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. Then, the spatial positions of the fluorescence spot of the probe on the DNA molecule were determined by image analysis.     

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.     

Single DNA molecule analysis: applications of molecular combing.

Dynamic changes to the genomic structure and to the DNA replication programme are important determinants of normal and abnormal cell development. To understand these changes and how they vary from cell to cell, single DNA molecules from both normal and abnormal cell populations must be examined and compared. Physical characterisation of single genomes at the kilobase level of resolution over large genomic regions is possible with molecular combing technology. An array of combed single DNA molecules is prepared by stretching molecules attached by their extremities to a silanised glass surface with a receding air-water meniscus. By performing fluorescent hybridisation on combed DNA, genomic probe position can be directly visualised, providing a means to construct physical maps and detect micro-rearrangements. Single-molecule DNA replication can also be monitored through fluorescent detection of incorporated nucleotide analogues on combed DNA molecules. These and other single-molecule applications of molecular combing are discussed in this paper and future developments of the technology are considered.     

Single molecule imaging reveals a slice of life

JGP study describes method to trace the real-time movements of individual membrane proteins in live tissue slices.

Directly observing the movements of single, fluorescently labeled molecules can provide crucial information about a molecule’s interactions in living cells. Plasma membrane proteins, for example, may freely diffuse around the lipid bilayer, pausing only when they collide and interact with other proteins. These movements can be followed relatively easily in single-cell organisms or cultured mammalian cells but are much more challenging to observe in multicellular organisms, where cell–cell interactions can dramatically alter the properties of the plasma membrane. In this issue of JGP, Mashanov et al. describe a new method to image and track individual plasma membrane proteins in living tissue slices (1).Justin Molloy (left), Gregory Mashanov (right), and colleagues describe a method to image single plasma membrane proteins in live tissue slices. By tracking individual M₂ muscarinic acetylcholine receptors in cardiac tissue over time, the researchers can construct a super-resolution map of the tissue, encompassing both the round cardiomyocytes and the ultrathin nerve fibers that innervate them.Justin Molloy’s group at The Francis Crick Institute in London are interested in how the M₂ muscarinic acetylcholine receptor regulates the heartbeat. This G protein–coupled receptor diffuses through the plasma membrane and, in response to acetylcholine, alters the resting potential of cardiomyocytes via a G_βγ-mediated interaction with inwardly rectifying potassium GIRK channels (2, 3).“It’s a diffusion-limited signaling cascade, so it’s important to look at the movement of the molecules within the membrane,” Molloy explains. “We’ve tracked the movements of single M₂ receptors in cultured cardiomyocytes, but we wanted to do it in tissues where the cells are in their native environment.”Molloy and colleagues, led by Gregory Mashanov, developed a technique to image single M₂ receptors in cardiac tissue slices (1). Freshly extracted mouse hearts are quickly placed in a custom-made, 3-D–printed cutting block, then sectioned by a multi-blade assembly into 1-mm-thick slices. These slices are treated with a fluorescently labeled ligand that tightly binds to M₂ receptors, before being transferred to coverslips for TIRF video microscopy.Mashanov immediately noticed that cardiomyocytes in living tissue are much more rounded than they are in cell culture. More remarkable still, however, were the differences Mashanov observed when he compared the movements of single M₂ receptors in cells and tissues. “The M₂ receptors move around the membrane around four times faster in tissue than they do in cultured cells,” Mashanov says.The reason for this increased mobility in tissues remains unclear, but Mashanov et al. saw a similarly rapid movement of M₂ receptors in zebrafish hearts, which the researchers were also able to dissect and prepare for TIRF microscopy with their new technique, even though these organs measure just ∼0.5 mm in length.In addition, the researchers discovered that they could use their single-molecule tracking data to create super-resolution images of the cardiac tissue slices. “When we average our tracking data over time, the paths of individual M₂ receptors combine to delineate the cellular structure of the tissue,” Molloy explains.Because neurons also express M₂ receptors, these super-resolution tissue maps include not only the cardiomyocytes but also the nerve fibers that innervate them. “These nerve fibers are only ∼0.2 μM in diameter and they aren’t really visible by light microscopy,” Mashanov says. “But we could see hundreds of them. Every cardiomyocyte has a nerve fiber associated with it.”Mashanov et al.’s technique should be easily adapted for other tissues and membrane proteins and may even facilitate single-molecule imaging in entire model organisms like zebrafish or fruit flies. For Molloy’s laboratory, though, the next step is to develop dual-color labeling of M₂ receptors and the downstream proteins in the pathway, G_βγ and GIRK, so that the kinetics of the molecules’ interactions can be studied in living tissues.     

Surface mutagenesis of the bovine papillomavirus E1 DNA binding domain reveals residues required for multiple functions related to DNA replication

The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.     

Repair of DNA in replicated and unreplicated portions of the human genome.

Portions of the human genome that have replicated after ultraviolet light irradiation and those that remain unreplicated have both been examined for the distribution of pyrimidine dimers and the extent of repair replication following their removal. The data indicate that the number of unrepaired dimers and the extent of repair replication seen after their excision are equal in the replicated and unreplicated DNA. Furthermore, the daughter strand of replicated DNA is larger than the average interdimer distance found in the parental strand. Hence, DNA replication in normal human fibroblasts is clearly capable of getting past pyrimidine dimers, and a preferential repair of such lesions in DNA that is about to be or has been replicated does not operate to any visible extent in these cells.     

Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.     

NSMF promotes the replication stress-induced DNA damage response for genome maintenance

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.     

Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variation

Necator americanus is a blood-sucking, intestinal nematode of major human health importance in many tropical and subtropical regions of the world. The aim of the present study was to compare the complete mitochondrial genome sequence from one N. americanus individual from Togo with another from China, in order to estimate the magnitude of genetic variability for different mitochondrial genes and non-coding regions. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (3-7%) and amino acid (1-7%) levels. The most conserved of these was the nad4L gene, whereas the nad1 gene was least conserved at both the nucleotide and amino acid levels. Nucleotide differences were also detected in 14 of the 22 transfer RNAs (trns) (1-13%), the AT-rich region ( approximately 8%), non-coding regions (8-25%) and in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rrn) ( approximately 1%). Comparison of the rrnL sequences among multiple individual worms revealed nine unequivocal nucleotide differences between N. americanus from the two countries. Consistent with previous studies, these findings provide evidence for substantial genetic variation within N. americanus, which may have implications for the transmission and control of hookworm disease.     

Single molecule analysis of the actomyosin motor

Progress in imaging techniques and nano-manipulation of single molecules has been remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and of how the mechanical cycles of the actomyosin motor are coupled to ATP hydrolysis. This has been achieved by measuring mechanical and chemical events of actomyosin directly at the single molecule level. Recent studies have made detailed measurements of myosin step size and mechanochemical coupling. The results of these studies suggest a new model for the mechanism of motion underlying actomyosin motors, which differs from the currently accepted lever-arm swinging model.