The DNA Sliding Clamp

In unicellular eukaryotes (kinetoplastids), two distinct PCNA are operational. Toxoplasma gondii separates an exclusively intranuclear, and a both intranuclear and a cytoplasmic PCNA in the S phase, both PCNAs serve inside the nucleus. In Plasmodium falciparum, one PCNA interacts with the original recognition complex (ORCs) of S. cerevisiae homologue, and co-localizes with it within the nucleus [1072]. The Trypanosoma brucei pre-RC protein is a Cdc45, which interacts with a 

The central nervous system of the ascidian tadpole larvae consist of 370 cells with PCNA proteins in the nuclei. An antisense oligonucleotide can inhibit the PCNA mRNA. Inhibition of PCNA resulted in deformed head development with cessation of DNA synthesis and nuclear DNA fragmentation resembling programmed cell death [1094]. The drosophila or mosquito dacapo is a Cip/Kip family cell cycle inhibitoiy protein of 261aa residues (cycline-dependent kinase inhibitory protein kinase inhibitory protein). The serine-threo/any aa/glutamate/aspartic acid 

MAPK/ERK pathway [1107], but the connection, if any, betweeen PCNA and ERK remains to be investigated. 

In the sea urchin embryo, the ERK pathway drives PCNA activity, and ERK inhibitors switch off PCNA-driven DNA replication [1108] vide supra). In human cells, as well as in other eukaryotes, small ubiquitin-Uke modifiers (SUMO) sumoylate PCNA at multiple sites. PCNA-SUMOl fusion prevents dsDNA breaks and recombinations due replication fork collapse at stalling DN A lesion sites [ 1109]. 

Comment. A key actor of DNA replication (PCNA) appeared at the dawn of life in the archaea (before the cell nucleus developed, or was acquired), and then in the unicellular eukaryotes to sustain their DNA rephcation (error-prone, or error-free). Its pathway constitutively reappears in the mahgnantly transformed cells of the mammalian hosts, providing them with an irreversible stimulus to sustain their hfe, disorganized as it may be, but nevertheless, immortalized. 

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DNA polymerase III can polymerize DNA, but with a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the addition of the J8 subunits, four of which complete the DNA polymerase III holoenzyme. The J3 subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps (Fig. 25-10b). Each dimer associates with a core subassembly of polymerase III (one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds. The J8 sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity—to greater than 500,000 (Table 25-1). 

The key features of DNA polymerase III are its catalytic potency, its fidelity, and its processivity. It is able to catalyze the addition of 10 bases per second, compared to only about 10 per second for DNA polymerase I, with an error frequency of about 1 10 -10. One of the unique features of DNA polymerase III is its ability to continuously synthesize very long (thousands of bases) stretches of DNA, unlike DNA polymerase I. This is a consequence of its processivity the P2 sliding clamps ensure that it remains bound to the template strand DNA. In contrast, DNA polymerase I, without a sliding clamp, is much more likely to dissociate from the template strand after synthesis of short (tens of bases) stretches of DNA. 

Lopez de Saro, F. J., and O Donnell, M. (2001). Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I. Proc. Natl. Acad. Sci. USA 98, 8376-8380. 

Family C of DNA polymerases is presented exceptionally by bacterial enzymes involved in replicative processes. So, the DNA polymerase III holoenzyme is the main enzyme realizing the DNA replication in Escherichia coli. Bacillus subtilis, and belongs to family C polymerases. It consists of three assemblies the pol 111 core, the beta sliding clamp processivity factor and the clamp-loading complex. The core consists of three subunits—a, the polymerase activity hub, 8, exonucleolytic proofreader, and 0, which may act as a stabilizer for e. The holoenzyme contains two cores, one for each strand, the lagging and leading [19]. The beta shding clamp processivity factor is also present in duplicate, one for each core, to create a clamp... 

Kong, X-P, R. Onrust, M. O Donnell, and J. Kuriyan, Three-dimensional structure of the /3 subunit of E. coli DNA polymerase HI holoenzyme A sliding clamp. Cell 69 425-437, 1992. 

Indian C, Mclnerney P, Georgescu R, Goodman ME, O Donnell M. A sliding-clamp toolbelt binds high- and low-fidelity DNA polymerases simultaneously. Mol. Cell 2005 19 805-815. Bunting KA, Roe SM, Pearl LH. Structural basis for recruitment of translesion DNA polymerase Pol JV/DinB to the beta-clamp. Embo. J. 2003 22 5883-5892. 

Majka J, Burgers PM. Yeast Radl7/Mec3/Ddcl a sliding clamp for the DNA damage checkpoint. Proc. Natl. Acad. Sci. U.S.A. 2003 100(5) 2249-2254. 

Figure 27.31. Structure of the Sliding Clamp. The dimeric P2 subunit of DNA polymerase III forms a ring that surrounds the DNA duplex. It allows the polymerase enzyme to move without falling off the DNA substrate. 

The polymerase holoenzyme assembles. The DNA polymerase ITT holoen-zyme assembles on the prepriming complex, initiated by interactions between DnaB and the sliding clamp subunit of DNA polymerase 111. These interactions also trigger ATP hydrolysis within the DnaA subunits, signaling the initiation of DNA replication. The breakup of the DnaA assembly prevents additional rounds of replication from beginning at the replication origin. 

The second functional difference between the DNA polymerases is the level of processivity, which refers to the number of nucleotides added before the polymerase dissociates from the template. Again, DNA pol III is the clear winner in this category, as might be expected based on its central role in DNA replication. The b subunit of DNA pol III is designated as the processivity subunit. Two b subunits form a "clamp" around the template strand, which then can slide along the DNA (Fig. 22.5). This arrangement ensures that an active holoenzyme (all the DNA pol III subunits combined) remains tightly associated with DNA to enhance processivity. 

Sliding clamp - a protein dimer that encircles the DNA strand and helps hold the DNA polymerase to the DNA strand. 

Functions as a sliding clamp to hold the holoenzyme complex to DNA, making the enzyme very processive. 

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