The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. T he replication of DNA in eubacteria involves many proteins organized into a complex multifunctional machine termed the replisome. A central enzyme is the multisubunit DNA polymerase III holoenzyme. In Escherichia coli, and probably in most other eubacteria, the DnaE ortholog (␣ subunit) is in the core of the replicative polymerase, whereas in many Grampositive organisms a related enzyme, PolC, is proposed to have this function (1). The processivity of the polymerase is conferred by the direct interaction of the ␤ subunit (clamp protein) of DNA polymerase III (2, 3), with the DnaE (and presumably PolC) subunits. ␤ is loaded onto DNA by a clamp loader comprised of single ␦ and ␦Ј subunits and four ͞␥ subunits (1). The ␤ dimer thence encircles the DNA without actually binding to it. In addition to DnaE, three other E. coli DNA polymerases appear to interact with ␤. PolB (DNA polymerase II) is involved in DNA repair (4) and the addition of ␤ and the clamp loader increases its processivity in vitro (5, 6). Similarly, ␤ and the clamp loader together increase both the processivity (7) and efficiency (8) of DNA synthesis by DNA polymerase IV (DinB). ␤ also appears to play a similar role in the activity of DNA polymerase V (8) (the UmuDЈ 2 UmuC complex) and the UmuD subunit has been shown to bind to ␤ (9).Experimental evidence shows that at least some ␤-binding proteins can interact productively with ␤ from heterologous species. For example, PolC subunits from Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1, 10, 11). In contrast, E. coli DnaE cannot use ␤ from the other species (11), the E. coli clamp loader complex cannot load S. aureus ␤ (11), and the S. pyogenes clamp loader complex cannot load E. coli ␤ (1).In the absence of any experimentally identified ␤-binding sites in proteins, a bioinformatics approach was undertaken to identify putative ␤-binding motifs. The role of the putative motif was then examined by yeast two-hybrid and peptide-binding experiments with native and modified sequences. Materials and MethodsSources of Amino Acid Sequences. Amino acid sequences and alignments were derived from: PSI-BLAST of the protein database at the National Center for Biotechnology Information (NCBI), and BLAST of preliminary sequence data from NCBI at http:͞͞ www.ncbi.nlm.nih.gov͞Microb_blast͞unfinishedgenome.html, Institute for Genomic Research at http:͞͞www.tigr.org, Department of Energy Joint Genome Institute at http:͞͞spider.jgipsf.org͞JGI_microbial͞html͞, Sanger Center at http:͞͞ www.sanger.ac.uk͞DataSearch͞omniblast.shtml, and ERGO at http:͞͞wit.integratedgenomics.com͞IGwit͞. Alignments of available sequences of all members of eubacterial protein families known to bind to ␤ were compiled with manual editing in regions of variab...

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The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.

In Escherichia coli, interactions between the replication initiation protein DnaA, the ␤ subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. DNA replication consists of three sequential steps: initiation, elongation, and termination. In Escherichia coli the initiation of a new round of chromosome replication occurs when the initiation protein, DnaA, binds to a 9-mer DnaA box within the chromosomal origin, oriC (reviewed in references 34 and 41). In vitro studies have shown that the binding of DnaA to oriC in the presence of the DNA structural protein HU or IHF (14,16,40) stimulates opening of the DNA duplex by melting the AT-rich 13-mer region in oriC (4,9,26). The unwound region then provides an entry site for the DnaB-DnaC helicase, which expands the region of single-stranded DNA. DnaG primase, single-stranded DNA-binding protein, DNA polymerase III holoenzyme (Pol III), and other proteins required for the replication fork formation are then recruited, and bidirectional DNA synthesis is initiated (13).In normal growth, cells replicate their DNA once before cell division and in E. coli there are at least three different mechanisms that block the occurrence of multiple initiations. The

The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The β(2) sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in β(2) by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-β(2) interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to β(2), as a starting point for further inhibitor design.

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