In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments 1,2 . At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leadingstrand polymerase. The replication loop grows and shrinks during each cycle of Okazaki-fragment synthesis 3 . Here, we employ single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.The 'trombone model' as proposed by Bruce Alberts 3 provides an elegant model for coupling many rounds of Okazaki-fragment synthesis on the lagging strand of the replication fork to the continuous production of DNA on the leading strand. The looping back of the lagging strand onto the replisome allows both leading-and lagging-strand DNA polymerases to synthesize in the same direction and facilitates recycling of the lagging-strand DNA polymerase by virtue of its proximity to the RNA primers newly synthesized at the fork. The visualization of replication intermediates of the T4 and T7 bacteriophage replication systems by electron microscopy (EM) demonstrated the existence of replication loops and allowed a characterization of their length distributions 4,5 . Biochemical studies revealed a number of molecular scenarios that explain how formation and release of replication loops may be regulated 6-14 . However, no dynamic characterization has been reported and the timeline controlling the various enzymatic activities at the fork is not entirely understood.In this study, we report the reconstitution of T7 replisomes and the observation of coordinated leading-and lagging-strand synthesis at the single-molecule level. The T7 replisome consists of only four proteins and it displays all the important features of more complicated replication systems 15 (Fig. 1a). These proteins are: the T7 DNA polymerase, a 1:1 complex of T7 gene 5 protein (gp5) and Escherichia coli thioredoxin processivity factor; T7 gene 4 helicase/primase protein (gp4); and T7 gene 2.5 single-stranded DNA binding protein (gp2.5). In previous work, we used single-molecule techniques to study the activity of partiall...

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading-and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.processivity ͉ replisome ͉ gp5-gp4 ͉ dynamic T he replisome (Fig. 1) of bacteriophage T7 consists of three phage-encoded proteins and one host protein (1). DNA synthesis is carried out by the gene 5 DNA polymerase (gp5) after forming a tight one-to-one complex with Escherichia coli thioredoxin (trx), the processivity factor (2, 3). The multifunctional gene 4 protein (gp4) provides both primase and helicase activities (4-7), and the gene 2.5 protein (gp2.5) functions as a ssDNA-binding protein (8). Although the size of the T7 replisome is modest, the multiple interactions of the four proteins give rise to functions provided by additional proteins in other systems. For example, a unique insert in the thumb of gp5, the thioredoxin-binding domain (TBD), provides a binding site for the processivity factor (9-11) and for the binding of gp4 and gp2.5 (11). Furthermore, the covalent attachment of the primase to the helicase in gp4 provides for the requisite interaction of these two components of the replisome. Accessory proteins such as clamp and helicase loaders are not required in the T7 system. Nonetheless, leading-and lagging-strand syntheses are processive and coordinated (12). The lagging strand is synthesized in a discontinuous manner with nascent Okazaki fragments located in replication loops (12,13).A mechanism to coordinate leading-and lagging-strand syntheses is dictated by the inherently more complex events that occur on the lagging strand. Whereas the leading strand can be synthesized in an uninterrupted fashion, lagging-strand synthesis must occur through the synthesis of short Okazaki fragments (Fig. 1), an event that necessitates the synthesis of an RNA primer...

BackgroundThe adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.Principal FindingsWe found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.ConclusionsThese results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.

Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes.