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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumInteraction of the β sliding clamp with MutS, ligase, and DNA polymerase IFrancisco J.López de Saro* and Mike O’Donnell Howard Hughes Medical Institute and The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10021 The β 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 Esche richia coli β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the β clamp. Thus, β 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 β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication. PCNA | mismatch repair The Sliding Clamp in ReplicationThe highly processive DNA polymerases that replicate cellular genomes combine a tight grip on DNA with rapid mobility along the duplex during synthesis (reviewed in refs. 1 and 2). The apparent contradiction of binding to DNA tightly, yet rapidly moving on DNA, is resolved by a mechanism that on hindsight is quite elegant and simple. The solution lies in a protein molecule in the shape of a ring that, rather than binding to DNA by using direct chemical forces, binds to DNA by encircling it (i.e., topological binding). This mechanism was first discovered in the Escherichia coli system in which the β dimer, a subunit of DNA polymerase III holoenzyme (Pol III H.E.), was found to be able to bind tightly to circular DNA but to slide off the ends of linear DNA (3). The dependence of the beta-DNA interaction on the topological state of DNA indicated that the protein does not bind DNA through the usual chemical forces, but instead binds DNA by virtue of its shape (i.e., as a ring, or clamp). The β subunit therefore serves as a mobile sliding clamp to continuously tether Pol III H.E. to DNA during synthesis, thus assuring its processivity. Crystal structure analysis of E. coli β and its functional homologues, eukaryotic proliferating cell nuclear antigen (PCNA) and phage T4 gp45, has revealed the ring shape of all of these “processivity factors” (Fig. 1 A-C; refs. 4–7). The fold of the polypeptide chain in β and PCNA is essentially the same, which is remarkable given the fact that no homology is observed between the two proteins. The chain fold of gp45 is similar, with the exception of the loss of two secondary structural elements. The overall structures of the homoligomeric β and PCNA rings can be described as being composed of six globular domains, each of the same shape, which pack together to form a six domain ring. The architecture of the ring includes a continuous layer of pleated sheet structure around the entire circumference, and a set of 12 α-helices that line the inside of the ring. A major difference between β, PCNA, and gp45 is their oligomeric composition. β is a homodimer in which each monomer consists of three domains; PCNA and gp45 monomers consist of only two domains and trimerize to form the ring. Sliding clamps require a clamp loader assembly for their topological association with DNA. Clamp loaders consist of multiple subunits and require ATP to perform their task as a “molecular matchmaker” between the sliding clamp and the DNA (reviewed in refs. 1 and 8). The workings of the E. coli clamp loader, called the γ complex, will be discussed later in this report. After assembly of the sliding clamp onto DNA, the polymerase associates with the same side of the ring used by the clamp loader (see scheme in Fig. 1D). PCNA and gp45 Participate in Many DNA Metabolic ProcessesIn this report, we demonstrate that β interacts with other proteins besides those directly involved in chromosomal replication. First, however, it seems prudent to briefly review what is known in this regard in other systems, especially considering the broad scope of proteins that are known to interact with PCNA. Although the clamps were first discovered for their use in replication, it soon became apparent that they are also involved in many other DNA metabolic processes. The first example of this was in the T4 system in which Geiduschek and coworkers demonstrated that the T4 gp45 clamp can act as a “mobile enhancer” to activate transcription of phage late genes (9). This work demonstrated that T4 gp45 assembles onto DNA at a nicked site and interacts with phage-modified E. coli RNA polymerase, which is then active for specific transcription from the phage late-gene promoters. PCNA is known to function with DNA polymerases δ and ε, enzymes responsible for chromosome replication (2). The first indication that PCNA may act with a protein other than a DNA polymerase came from an observation of PCNA in complex with p21C1P1/WAF1, the cyclin kinase inhibitor (10). This interaction is thought to play a regulatory role in replication because p21 blocks use of PCNA by Pol δ (11, 12). Since this observation was made, several other proteins have been shown to interact with PCNA (reviewed in ref. 10). These include XPG, MSH3, MSH6, MLH1, PMS2, and hMYH (13), implying roles for PCNA in nucleotide excision repair, mismatch repair, and base excision repair. PCNA also interacts with Fen1 endonuclease and DNA ligase I, suggesting a role in the maturation of Okazaki fragments or perhaps other processes involving the trimming of 5′ ends or filling of DNA gaps. PCNA also binds the chromatin assembly factor 1 (CAF-1; ref. 14), and several other proteins, including a DNA methyltransferase and gadd45. This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: Pol III H.E., DNA polymerase III holoenzyme; PCNA, proliferating cell nuclear antigen.
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