The E. coli replication machinery employs a beta clamp that tethers the polymerase to DNA, thus ensuring high processivity. The replicase also contains a processivity switch that dissociates the polymerase from its beta clamp. The switch requires the tau subunit of the clamp loader and is regulated by different DNA structures. At a primed site, the switch is "off." When the replicase reaches the downstream primer to form a nick, the switch is flipped, and tau ejects the polymerase from beta. This switch has high fidelity for completed synthesis, remaining "off" until just prior to incorporation of the last nucleotide and turning "on" only after addition of the last dNTP. These actions of tau are confined to its C-terminal region, which is located outside the clamp loading apparatus. Thus, this highly processive replication machine has evolved a mechanism to specifically counteract processivity at a defined time in the lagging-strand cycle.
In Escherichia coli, the circular  sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from  and DNA and cycles to a new start site, a primed template loaded with . DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t1 ⁄2 ϳ 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that ␦, a single subunit of DNA polymerase III holoenzyme, opens  and slips it off DNA (k unloading ؍ 0.011 s ؊1 ) at a rate similar to that of the multisubunit ␥ complex clamp loader by itself (0.015 s ؊1 ) or within polymerase (pol) III* (0.0065 s ؊1 ). Moreover, unlike ␥ complex and pol III*, ␦ does not require ATP to catalyze clamp unloading. Quantitation of ␥ complex subunits (␥, ␦, ␦, , ) in E. coli cells reveals an excess of ␦, free from ␥ complex and pol III*. Since pol III* and ␥ complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the ␦ subunit probably aids  clamp recycling during DNA replication.Sliding clamps are ring-shaped proteins that completely encircle DNA and slide freely along the double helix. This unique topological linkage between protein and DNA allows the clamp to tether DNA polymerase to the template and move along with the polymerase as it extends a new DNA strand (1). Thus, the circular clamp serves as a processivity factor, allowing DNA polymerase to replicate several thousand nucleotides without dissociation from the template (2, 3).The DNA polymerase (pol)
The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ringshaped protein, the  clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The  clamp is assembled onto DNA by the multiprotein ␥ complex clamp loader that opens and closes the  ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the ␥ complex assembles  onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the ␥ complex does not assemble  onto relaxed closed circular DNA even at low ionic strength. Hence, a 3-end is not required for clamp loading, but a singlestranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the ␥ complex exhibits polarity; it acts specifically at the 3-ssDNA/dsDNA junction to assemble  onto the DNA. The ␥ complex can assemble  onto a primed site as short as 10 nucleotides, corresponding to the width of the  ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3 terminus prevents the clamp loading reaction, indicating that the ␥ complex and its associated  clamp interact with ϳ14 -16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20 -22 bp from the 3 terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the ␥ complex on the duplex portion of the primed site for function with . Interestingly, DNA secondary structure elements placed near the 3 terminus impose similar steric limits on the ␥ complex and polymerase action with . The possible biological significance of these structural constraints is discussed.Escherichia coli DNA polymerase III holoenzyme (pol III 1 holoenzyme) is a highly processive multisubunit replicase (1). Processivity is conferred to the polymerase by the  subunit (2, 3). The  subunit is a ring-shaped protein that completely encircles DNA and slides along the duplex (4, 5). The  ring endows the polymerase with high processivity by binding directly to it, continuously tethering it to the template during synthesis (4). The  ring does not assemble onto DNA by itself; for this, it requires the clamp loading action of the ␥ complex. The ␥ complex is composed of five different proteins (␥, ␦, ␦Ј, , and ) (1). Upon binding ATP, the ␥ complex opens the  ring and positions it around the primed template (7,8). Hydrolysis of two molecules of ATP results in closing the ring around DNA and dissociation of the ␥ complex from the clamp (7, 49).Following the release of the clamp loader from the clamp, the catalytic core polymerase subassembly (pol ...
The Escherichia coli  dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The ␥ complex (␥␦␦) clamp loader couples ATP to the opening and closing of  in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The ␦ subunit of the E. coli ␥ complex clamp loader is known to bind  and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of ␥ complex also bind , although weaker than ␦. The ␥ subunit like ␦, affects the opening of , but with a lower efficiency than ␦. The ␦ subunit regulates both ␥ and ␦ ring opening activities in a fashion that is modulated by ATP interaction with ␥. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.Chromosomal replicases are highly processive machines owing to a sliding clamp subunit that encircles and slides on DNA, acting as a mobile tether for the replicase during synthesis (1-4). These circular clamps require a multimeric clamp loader assembly for their opening and closure around DNA in a process that consumes ATP. In Escherichia coli the clamp is the  dimer, formed from two crescent-shaped protomers (5), and the  ring is opened and closed by the ␥ complex clamp loader (␥␦␦Ј). Once on DNA,  acts as a mobile tether for the replicase, DNA polymerase III holoenzyme, holding it to DNA for highly processive synthesis (1). In fact, the  subunit can also couple with all the other E. coli DNA polymerases (DNA polymerases I, II, IV, and V) (6 -9) and with DNA ligase and MutS (10). The eukaryotic system is similar (11). Here, the RFC 1 clamp loader assembles the ring-shaped PCNA clamp onto DNA for processive DNA polymerase action (12, 13). PCNA is also known to interact with several other proteins indicating that, like , these clamps serve multiple roles in cellular DNA metabolism (14).This report is part of a continuing study on the mechanism of the E. coli ␥ complex clamp loader. The ␥ complex consists of five different subunits (␥␦␦Ј) (15), three of which (␥␦␦Ј) are essential to clamp loading action (16). One copy each of the and subunits bind the ␥␦␦Ј core but are not essential to clamp loading activity (17). The crystal structure of the ␥␦␦Ј complex has recently been solved, and it shows that there are three ␥ subunits and one each of ␦ and ␦Ј in a circular pentameric arrangement (18). A protein in the holoenzyme known as is encoded by the same gene as ␥ (dnaX) and therefore is essentially identical to ␥ except for an extra C-terminal section in (19,20). Fully active clamp loading complexes can be reconstituted and are composed of one each of ␦, ␦Ј, and either 3 or ␥ 3 , or mixtures of and ␥ (i.e. 1 ␥ 2 and 2 ␥ 1 ) (17, 21). The unique C terminus of , lacking in ␥, binds to the DNA pol...
The dual effect of the ubiquitous inflammatory cytokine transforming growth factor B1 (TGFB) on cellular proliferation and tumor metastasis is intriguing but complex. In epithelial cell -and neural cell -derived tumors, TGFB serves as a growth inhibitor at the beginning of tumor development but later becomes a growth accelerator for transformed tumors. The somatostatin (SST) signaling pathway is a well-established antiproliferation signal, and in this report, we explore the interplay between the SST and TGFB signaling pathways in the human neuroendocrine tumor cell line BON. We defined the SST signaling pathway as a determinant for neuroendocrine tumor BON cells in responding to TGFB as a growth inhibitor. We also determined that TGFB induces the production of SST and potentially activates the negative growth autocrine loop of SST, which leads to the downstream induction of multiple growth inhibitory effectors: protein tyrosine phosphatases (i.e., SHPTP1 and SHPTP2), p21Waf1/Cip1 , and p27 Kip1 . Concurrently, TGFB down-regulates the growth accelerator c-Myc protein and, collectively, they establish a firm antiproliferation effect on BON cells. Additionally, any disruption in the activation of either the TGFB or SST signaling pathway in BON leads to ''reversible'' neuroendocrinemesenchymal transition, which is characterized by the loss of neuroendocrine markers (i.e., chromogranin A and PGP 9.5), as well as the altered expression of mesenchymal proteins (i.e., elevated vimentin and Twist and decreased E-cadherin), which has previously been associated with elevated metastatic potential. In summary, TGFB-dependent growth inhibition and differentiation is mediated by the SST signaling pathway. Therefore, any disruption of this TGFB-SST connection allows BON cells to respond to TGFB as a growth accelerator instead of a growth suppressor. This model can potentially apply to other cell types that exhibit a similar interaction of these pathways.
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