The ␤ 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 ␤ clamp is known to function with DNA polymerases II and V, indicating that ␤ also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the ␤ clamp. Thus, ␤ 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 ␤ interactive proteins suggests that the prokaryotic ␤ ring participates in a wide variety of reactions beyond its role in chromosomal replication. PCNA ͉ mismatch repairThe Sliding Clamp in Replication

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 b-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the a polymerase subunit of DNA polymerase III interacts with the b-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 d-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 b 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.

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 ␤ 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 ␤ 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 ␤ locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with ␤ 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 ␤ 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 ␤ with MutS. We find that two distinct regions of MutS interact with ␤. 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 ␤ interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for ␤ is not. In light of these results, we propose roles for the ␤ clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.The process of chromosomal replication can generate small deletions, insertions, or mismatches in newly synthesized DNA. If left unrepaired, post-replicative DNA damage can lead to genomic instability and increased rates of mutation (1, 2). These types of replicative errors are corrected by a set of enzymes that are specialized in performing DNA mismatch repair (MMR) 2 and are conserved across all domains of life. In Escherichia coli it is estimated that the fidelity of replication is increased Ͼ100-fold by the action of MMR (3). In humans a deficiency in MMR has been linked to increased susceptibility to certain sporadic cancers (4 -6).The E. coli MutS protein is an essential component of MMR and it directly binds to abnormal DNA structures to initiate the cascade of events that lead to repair. These events include the removal of a segment of the newly synthesized DNA strand by the combined action of a helicase and one or more nucleases, and the re-synthesis of the resulting gap by a DNA polymerase (1). A second essential component of MMR in E. coli is MutL, which is thought to link the action of MutS with nucleases and a helicase (1, 7). However, although the individual components of MMR in prokaryotes and eukaryotes are mostly well described, many of the details of the repair mechanism are still poorly defined.In eukaryotic organisms...