Quality control in the initiation of eukaryotic DNA replication

Origins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.     

Emerging players in the initiation of eukaryotic DNA replication

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression.     

Emerging mechanisms of eukaryotic DNA replication initiation

Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45. Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes. One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a ‘clamp-loader’ required to lock the MCMs around DNA. The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins. Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.     

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.     

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.     

DNA polymerases that propagate the eukaryotic DNA replication fork

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.     

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.     

Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

Atomic force microscopy and chemical force microscopy of microbial cells

Over the past years, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of microbial cells with nanometer resolution, and under physiological conditions. Moreover, chemical force microscopy (CFM) and single-molecule force spectroscopy have enabled researchers to map chemical groups and receptors on cell surfaces, providing valuable insight into their structure-function relationships. Here, we present protocols for analyzing spores of the pathogen Aspergillus fumigatus using real-time AFM imaging and CFM. We emphasize the use of porous polymer membranes for immobilizing single live cells, and the modification of gold-coated tips with alkanethiols for CFM measurements. We also discuss recording conditions and data interpretation, and provide recommendations for reliable experiments. For well-trained AFM users, the entire protocol can be completed in 2-3 d.     

Atomic force microscopy study of DNA conformation in the presence of drugs

Binding of ligands to DNA gives rise to several relevant biological and biomedical effects. Here, through the use of atomic force microscopy (AFM), we studied the consequences of drug binding on the morphology of single DNA molecules. In particular, we quantitatively analyzed the effects of three different DNA-binding molecules (doxorubicin, ethidium bromide, and netropsin) that exert various pharmacologic and therapeutic effects. The results of this study show the consequences of intercalation and groove molecular binding on DNA conformation. These single-molecule measurements demonstrate morphological features that reflect the specific modes of drug–DNA interaction. This experimental approach may have implications in the design of therapeutically effective agents.     

Atomic force microscopy studies on cellular elastic and viscoelastic properties

In this work, a method based on atomic force microscopy (AFM) approach-reside-retract experiments was established to simultaneously quantify the elastic and viscoelastic properties of single cells. First, the elastic and viscoelastic properties of normal breast cells and cancerous breast cells were measured, showing significant differences in Young’s modulus and relaxation times between normal and cancerous breast cells. Remarkable differences in cellular topography between normal and cancerous breast cells were also revealed by AFM imaging. Next, the elastic and viscoelasitc properties of three other types of cell lines and primary normal B lymphocytes were measured; results demonstrated the potential of cellular viscoelastic properties in complementing cellular Young’s modulus for discerning different states of cells. This research provides a novel way to quantify the mechanical properties of cells by AFM, which allows investigation of the biomechanical behaviors of single cells from multiple aspects.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Atomic force microscopy imaging of DNA under macromolecular crowding conditions

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl 2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt.     

Atomic force microscopy of DNA molecules stretched by spin-coating technique

We have developed an effective approach to stretching DNA molecules with the flow of fluid generated by spin coating. Well-stretched A DNA molecules were observed using atomic force microscopy. Substrate properties sensitively affected the stretching behavior of DNA. Our experimental findings revealed that a mica surface treated with crystal violet, a cationic dye molecule, is suitable to the spin-coating procedure for stretching DNA. Moreover, compared with relaxed DNA, we observed reduced height of the stretched DNA, which was attributed mainly to elongation force applied to the DNA molecules from the fluid flow and strong adhesion force between DNA and the substrate. This simple and effective method for preparing stretched DNA could be useful in physically mapping genomic DNA in a high throughput.     

Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation

The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.