Abstract: The molecular machines that replicate the genome consist of many interacting components. Essential to the organization of the replication machinery are ring-shaped proteins, like PCNA (Proliferating Cell Nuclear Antigen) or the β-clamp, collectively named sliding clamps. They encircle the DNA molecule and slide on it freely and bidirectionally. Sliding clamps are typically associated to DNA polymerases and provide these enzymes with the processivity required to synthesize large chromosomes. Additionally, they interact with a large array of proteins that perform enzymatic reactions on DNA, targeting and orchestrating their functions. In recent years there have been a large number of studies that have analyzed the structural details of how sliding clamps interact with their ligands. However, much remains to be learned in relation to how these interactions are regulated to occur coordinately and sequentially. Since sliding clamps participate in reactions in which many different enzymes bind and then release from the clamp in an orchestrated way, it is critical to analyze how these changes in affinity take place. In this review I focus the attention on the mechanisms by which various types of enzymes interact with sliding clamps and what is known about the regulation of this binding. Especially I describe emerging paradigms on how enzymes switch places on sliding clamps during DNA replication and repair of prokaryotic and eukaryotic genomes.

Abstract: DNA polymerase mu (Polmu) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polmu can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polmu, we have identified a specific DNA ligand residue (Arg(387)) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polmu mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg(387) in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polmu switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5'-P at a second DNA end, whose 3'-protrusion could provide a templating base to facilitate such a special "pre-catalytic translocation step." These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polmu could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.

Abstract: The MutL and MutS proteins are the central components of the DNA repair machinery that corrects mismatches generated by DNA polymerases during synthesis. We find that MutL interacts directly with the beta sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with beta only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on beta locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with beta without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the beta interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of beta with MutS. We find that two distinct regions of MutS interact with beta. One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal beta interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for beta is not. In light of these results, we propose roles for the beta clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.

Abstract: The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the beta clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.

Abstract: Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment. The Escherichia coli replicase contains a subunit (tau) that promotes separation of polymerase from its clamp on finishing DNA segments. This report reveals the mechanism of this process. We find that tau binds the C-terminal residues of the DNA polymerase. Surprisingly, this same C-terminal "tail" of the polymerase interacts with the beta clamp, and tau competes with beta for this sequence. Moreover, tau acts as a DNA sensor. On binding primed DNA, tau releases the polymerase tail, allowing polymerase to bind beta for processive synthesis. But on sensing the DNA is complete (duplex), tau sequesters the polymerase tail from beta, disengaging polymerase from DNA. Therefore, DNA sensing by tau switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.

Abstract: Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.

Abstract: The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the beta clamp. Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication.

Abstract: The delta protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme. In the presence of delta, RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling. Despite these profound effects, a strain containing a deletion of the delta gene (rpoE) is viable and shows no major alterations in gene expression. Quantitative immunoblotting experiments demonstrate that delta is present in molar excess relative to RNAP in both vegetative cells and spores. Expression of rpoE initiates from a single, sigmaA-dependent promoter and is maximal in transition phase. A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase. For biochemical analyses we have created derivatives of delta and sigmaA that can be radiolabeled with protein kinase A. Using electrophoretic mobility shift assays, we demonstrate that delta binds core RNAP with an apparent affinity of 2.5 x 10(6) M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta, and sigmaA.

Abstract: Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.

Abstract: RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.

Abstract: RNA polymerase purified from Bacillus subtilis is a complex mixture comprising a common core, the 20.4 kDa delta protein, and one of several sigma specificity factors. The delta protein, together with several truncated variants, was overproduced and purified from Escherichia coli. Delat is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate rich carboxyl-terminal region. The purified amino-terminal domain (deltaN) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (deltaC) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta but not sigma-a or deltaN displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to efficiently displace RNA requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase. Despite these profound effects on transcription, a strain containing a deletion of the delta gene (rpoE) is viable and the resulting phenotype shows no major alterations in the patterns of gene expression in the cell. I also report studies on the expression of the rpoE gene, the phenotype of the mutant and the abundance of this protein in the cell. Delta protein is approximately as abundant as RNAP (around 2,000 molecules per cell). A rpoE mutant is impaired in adjusting gene expression to shifts in growing conditions. I also show that delta binds RNAP specifically and that its binding affinity is comparable to that of other transcription factors. Finally, I provide evidence that delta is not present in the initiation complex at the promoter, as other studies had suggested.