Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes

An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA. Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro. Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro. Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo. Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication. Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.     

Sequence requirements for protein-primed initiation and elongation of phage O29 DNA replication

The double-stranded linear DNA of Bacillus subtilis phage O29 is replicated by a mechanism in which a terminal protein (TP) acts as a primer. The second 3'-terminal nucleotide of the template directs the incorporation of the 5'-terminal nucleotide into the TP, giving rise to the initiation complex TP-dAMP. Elongation then proceeds by a sliding-back mechanism in which the dAMP covalently linked to the TP pairs to the 3'-terminal nucleotide of the template strand to recover full-length DNA. We have studied the sequence requirements for efficient initiation of replication using mutated TP-free double-stranded DNA fragments. Efficient initiation only requires the terminal repetition 5'-AA. The 3'-terminal T, although not used as template, increases the affinity of DNA polymerase for the initiator nucleotide; in addition, although to a minor extent, the third 3'-terminal position also directs the formation of the initiation complex and modulates the initiation rate at the second position. Efficient elongation requires a previous sliding-back, demanding again a repetition of two nucleotides at the 3' end; if the sliding-back is prevented, a residual elongation can proceed directly from the second position or after jumping back from the third to the first position.     

The inhibition of both initiation and elongation of adenovirus DNA replication by actinomycin D

The effect of actinomycin D on adenovirus DNA replication has been examined both in vivo and in a cell-free extract capable of replication on exogenously added template. In both cases we show that 5 micrograms/ml of drug cause an inhibition of DNA synthesis of at least 80%. The in vitro results further demonstrate that both DNA chain growth (elongation) and initiation - the addition of the first nucleotide of the DNA chain (dCMP) to the preterminal protein - are inhibited directly by the drug, by not by alpha-amanitin.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses

To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.     

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.     

DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes.     

Cell-cycle-specific initiation of replication

The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle: the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle. Several fundamentally different methods have been used to study these processes: Meselson—Stahl density-shift experiments, experiments with the so-called‘baby machine', sorting of cells according to size, and flow cytometry. The evidence for precise timing and co-ordination of chromosome replication in Escherichia coli is overwhelming. Similarly, the high-copy-number plasmid ColE1 and the low-copy-number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle. Data about the low-copy-number plasmids F and P1 are conflicting. This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.     

In vitro replication of bacteriophage PRD1 DNA. Metal activation of protein-primed initiation and DNA elongation.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.     

Drosophila egg chamber elongation

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

Clamp loaders and replication initiation

Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic sources have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function.     

Dynamics of human replication factors in the elongation phase of DNA replication

In eukaryotic cells, DNA replication is carried out by coordinated actions of many proteins, including DNA polymerase δ (pol δ), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A. Here we describe dynamic properties of these proteins in the elongation step on a single-stranded M13 template, providing evidence that pol δ has a distributive nature over the 7 kb of the M13 template, repeating a frequent dissociation–association cycle at growing 3′-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol δ has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol δ has detached, or reload PCNA from solution to restart DNA synthesis. Furthermore, we suggest that a subunit of pol δ, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation–association cycles of pol δ. Based on these observations, we propose a model for dynamic processes in elongation complexes.     

Visualization of proprioceptors in Drosophila larvae and pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs). Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis. The development of larval ChOs during embryogenesis has been studied extensively. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy. In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary. Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.     

Chromosomal replication in Drosophila virilis

About half of the diploid genome of D. virilis is -heterochromatic (Heitz, 1934) and contains the satellite sequences found in isopycnic CsCl density gradients (Gall et al, 1971; Steinemann, 1976). The thymidine incorporation behavior of this material in the course of S phase was monitored by autoradiography. Labelled interphase nuclei show three types of labelling patterns, label exclusively confined to either eu- or -heterochromatin, and simultaneous labelling of both fractions. Using the fraction of labelled mitotic index method, the duration of the DNA-synthetic period, t_s = 11.9 ± 4.3 h and G₂ period, t_{G2 + 1/2M} = 6.9 ± 3.8 h, were determined. On the assumption that the investigated brain cells belong to an exponentially growing cell population, the cell cycle is 22.9 h long and the G₁ period lasts t_G1=4.1 h. The a-heterochromatin begins to replicate later than euchromatin and continues alone after a phase of common replication of both fractions. Noteworthy is the asynchronous termination in the proximal -heterochromatic segments of different chromosomes. Within the S phase, the first 1 h of DNA replication is exclusively confined to euchromatin, followed by 8 h of replication in both eu- and -heterochromatin and terminated by 3 h of exclusive -heterochromatin replication. Thus euchromatin has a doubling time of about 9 h and -heterochromatin of about 11 h. The -heterochromatin of D. virilis is late and slow replicating.