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...

No abstract

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.