Portal de Periódicos da CAPES

Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês

10.1093/emboj/cdg603

1460-2075

Francisco J. López de Saro,

Carcinogens and Genotoxicity Assessment

Article1 December 2003free access Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair Francisco J. López de Saro Francisco J. López de Saro Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Roxana E. Georgescu Roxana E. Georgescu Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Myron F. Goodman Myron F. Goodman Departments of Biological Science and Chemistry, University of Southern California, Los Angeles, CA, 90089 USA Search for more papers by this author Mike O'Donnell Corresponding Author Mike O'Donnell Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Francisco J. López de Saro Francisco J. López de Saro Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Roxana E. Georgescu Roxana E. Georgescu Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Myron F. Goodman Myron F. Goodman Departments of Biological Science and Chemistry, University of Southern California, Los Angeles, CA, 90089 USA Search for more papers by this author Mike O'Donnell Corresponding Author Mike O'Donnell Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Author Information Francisco J. López de Saro1, Roxana E. Georgescu1, Myron F. Goodman2 and Mike O'Donnell 1 1Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA 2Departments of Biological Science and Chemistry, University of Southern California, Los Angeles, CA, 90089 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6408-6418https://doi.org/10.1093/emboj/cdg603 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 β-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the α polymerase subunit of DNA polymerase III interacts with the β-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 δ-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 β 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. Introduction Protein clamps have been implicated in all processes of DNA metabolism, including replication, transcription, and various pathways of DNA repair (Warbrick, 2000; Stucki et al., 2001). Their biological significance stems, first, from the fact that they are tightly bound to DNA in a topological rather than sequence-specific manner that allows them to slide freely on the nucleic acid (Kong et al., 1992; Stukenberg et al., 1991) and, secondly, from the fact that they can interact with a variety of enzymes to increase their association to DNA. Many proteins that bind to the eukaryotic clamp [proliferating cell antigen (PCNA)] contain a consensus motif (Qxx[I/L]xxFF) that often localizes to the extreme N- or C-terminal regions of the protein (Warbrick, 2000). Polymerases from bacteriophages T4 and RB69 have also been found to interact with their clamps via conserved C-terminal sequences (Berdis et al., 1996; Shamoo and Steitz, 1999). Recently, the Y-family polymerase Pol IV of Escherichia coli was also found to interact with the β-clamp via C-terminal residues (Lenne-Samuel et al., 2002). In the case of bacterial chromosomal replicases of the Pol C family, exemplified by DNA polymerase III of E.coli, crystal structure information is lacking and no clear map of the clamp-polymerase interaction has been available. The DNA polymerase III core of E.coli consists of three subunits: α (DNA polymerase), ϵ (3′–5′ proofreading nuclease) and θ. The α DNA polymerase subunit of the Pol III core is responsible for the interaction with β (Stukenberg et al., 1991). Deletion studies of the E.coli α-subunit have suggested that the β interaction site resides in an internal region of the α-protein, spanning residues 542–991 (α is 129 kDa or 1160 amino acids) (Kim and McHenry, 1996). More recently, and based on limited similarity with other β-binding proteins, Dalrymple et al. (2001) proposed a β binding consensus sequence of QLxLF and suggested that β binds at an internal sequence of α at position 918–926. While we cannot exclude a role of internal sequences in the binding to the β-clamp, we have recently performed quantitative binding and functional assays that demonstrate that α binds β mainly through a different site located at the C-terminal 20 residues, and that these residues are required for function of α with β (López de Saro et al., 2003). These C-terminal residues of α are also required for the τ switch, which acts on α to regulate polymerase processivity. In E.coli it has been shown that the β-clamp can associate with all five known DNA polymerases in the cell (Pol I–V) to increase their processivity of synthesis (Hughes et al., 1991; Bonner et al., 1992; Tang et al., 2000; Wagner et al., 2000; López de Saro and O'Donnell, 2001; Kobayashi et al., 2002). These polymerases belong to four different structural families, namely family A (Pol I), family B (Pol II), family C (Pol III) and the newly discovered Y family (Pol IV and V) (Braithwaite and Ito, 1993; Ohmori et al., 2001; Filée et al., 2002), which have very limited or no apparent sequence similarity between them. Stimulation of DNA synthesis by the β-clamp is dependent on the topological linkage of the clamp to DNA (Stukenberg et al., 1991). This process involves the transient opening of the protein ring catalyzed by the γ-complex clamp-loader (γ3δδ′χψ) in a reaction driven by ATP hydrolysis (reviewed in O'Donnell et al., 2001). Within the γ-complex, the δ-subunit binds β the tightest and is responsible for opening of the clamp (Turner et al, 1999). In combination with the other γ complex subunits, the primed DNA is placed into the open β-ring and then ATP is hydrolyzed, allowing the ring to close around the DNA. Study of the β-clamp interaction with the δ-subunit of the γ-complex and the α-subunit of the Pol III core shows that they compete for β, and thus likely bind β in the same, or nearly the same, location (Naktinis et al., 1996). During lagging strand synthesis, Pol III core rapidly dissociates from the clamp at the end of each completed Okazaki fragment and hops to a new β-clamp on the next RNA-primed site to be extended (O'Donnell, 1987). The β-clamp is left behind on the completed DNA fragment and is free to function with other β-interactive proteins (Stukenberg et al., 1994). These include proteins involved in Okazaki-fragment maturation (Pol I and ligase), replication-associated DNA repair (Pol I and II), mutation-prone lesion bypass (Pol IV and V), and mismatch repair (MutS). The elucidation of the molecular structure of the γ-complex (Jeruzalmi et al., 2001), and of the δ-subunit of the γ-complex bound to the β-clamp (Jeruzalmi et al., 2001), was the first step in understanding the molecular details of how a protein interacts with β. The main attachment site of δ to β is mediated by δ-residues Leu73 and Phe74, which are highly conserved among prokaryotic δ-subunits (Jeruzalmi et al., 2001). The site on β to which δ binds is a hydrophobic pocket and residues that define the hydrophobic pocket of β are conserved (Jeruzalmi et al., 2001). The most defined view of how a DNA polymerase interacts with its clamp is provided by the crystal structure of an 11-residue C-terminal peptide of phage RB69 DNA polymerase (a B-family polymerase) bound to its corresponding clamp, the gp45 protein (Shamoo and Steitz, 1999). The interface mainly consists of a hydrophobic pocket in the gp45 clamp, similar to that displayed by δ binding to β. The cocrystal structure of a C-terminal peptide of the cell regulator, p21WAF1, to the human PCNA clamp is also mediated by a hydrophobic pocket in PCNA (Gulbis et al., 1996). In all three systems the clamps have remarkably similar architecture, consisting of six domains organized on either a dimer (β) or trimer (PCNA, gp45), and interaction with the clamp takes place at equivalent locations between domains of the clamps. These similarities suggest that the basic mechanism by which proteins bind to sliding clamps is conserved across all domains of life. As a first step towards understanding how clamps are used by multiple proteins during DNA synthesis and repair, one aim of the present study is to map in detail the site of interaction of the E.coli replicative polymerase, the α-subunit of Pol III, with the β-clamp and to examine whether it binds the same site in β as the δ-subunit of the clamp loader. We find that the C-terminal peptide of the Pol III α subunit inhibits clamp loading by the γ-complex and binding to β by the δ-subunit, suggesting that the α·β and δ·β interactions do indeed occur at the same locus on the clamp. This observation provides the structural basis for the internal competition for clamps in the Pol III holoenzyme replicative machinery (Naktinis et al., 1996). Another goal of the present study is to determine whether the other E.coli polymerases also interact with β in the same locus as α and δ, and if the interaction is mediated by C-terminal residues. We find that DNA polymerase II (Pol II), despite having no sequence similarity with the Pol III α-subunit, also interacts with the β-clamp in the same location, via the C-terminal end of the protein. Pol IV also interacts with β via C-terminal residues, consistent with a previous study (Lenne-Samuel et al., 2002), and we find that it binds β in the same locale as α and Pol II. Furthermore, we find that the sites of interaction between the clamp and DNA polymerases I and V are the same as those of Pol II and Pol III, although these polymerases do not appear to utilize C-terminal residues for this interaction. Overall, we find that all E.coli polymerases interact at the same point on the clamp, namely the hydrophobic pocket to which the δ-subunit binds. The implications of these findings with respect to how these enzymes traffic on the β-clamp are discussed. Results Characterization of an α-subunit C-terminal peptide interaction with the β-clamp We have recently demonstrated that the C-terminal 20 residues of the α-subunit are required for functional interaction with β (López de Saro et al., 2003). To define the critical residues that contribute to the α-β interaction, we designed overlapping N-biotinylated peptides spanning the C-terminal region of α and assayed them for interaction with the β-clamp by attaching them to streptavidin-coated microplates and then probing them for ability to bind 32P-β. Radiolabeled β used in these studies contained a six-residue C-terminal extension that can be phosphorylated with protein kinase A and [32P]γ-ATP (Stukenberg et al., 1994; Kelman et al., 1995). We found that peptides corresponding to the C-terminal 20 amino acids interact with the β-clamp, as did a peptide lacking the last residue, but deletion of two residues from the C-terminus abolished the interaction (Figure 1A). Hence, the penultimate F-residue of the α-peptide is required for interaction with β. Figure 1.The extreme C-terminus of the α-subunit of DNA polymerase III interacts with the β-clamp. (A) Sequences probed by peptide analysis are shown as stippled boxes in the DnaE protein scheme, but only the analysis of the C-terminal peptides is shown. N-terminal biotinylated peptides were immobilized on streptavidin-coated 96-well plates and probed with 32P-β. It is estimated that 30 nmol of peptide is retained in each well. Peptide sequences used in the microplate assay on the right are shown as lines under the sequence of the C-terminus. Results of the assays are shown to the right. (B) Native polyacrylamide electrophoresis was used to separate a complex of 32P-β with biotinylated α1141–1160 bound to streptavidin. Lane 1 contained only 32P-β; the α1141–1160 peptide (220 nM) was added in lanes 3 and 4, and streptavidin (2.2 μM) in lanes 2 and 4, as indicated. (C) Gel shift assay of the radiolabeled β-clamp. Native polyacrylamide electrophoresis was performed to separate free 32P-β from 32P-β·core complexes as described in Materials and methods. Pol III core was added in lanes 2–6 and the complex was challenged with either BSA (lane 2), non-labeled E.coli β-clamp (lane 3), human PCNA (lane 4), phage T4 gp45 (lane 5) or α1141–1160 peptide (lane 6), as indicated above the gel. (D) DNA synthesis is inhibited by α C-terminal peptides. Reactions were performed using primed M13mp18 ssDNA, β, core and γ complex in the presence of 100 μM of each peptide as described in Materials and methods. Download figure Download PowerPoint Next, we used a protein shift assay (López de Saro and O'Donnell, 2001) to determine whether the C-terminal α-peptide (α1141–1160) complexed to streptavidin in solution is sufficient to bind to 32P-β and to produce a mobility shift in a native polyacrylamide gel. The results showed that streptavidin does indeed produce a slower-mobility complex, presumably consisting of streptavidin·biotin-peptide bound to 32P-β (Figure 1B, lane 4). We also observed that Pol III core retards the mobility of 32P-β in a native gel (Figure 1C, compare lanes 1 and 2). If the polymerase·β interaction requires the α C-terminal sequences, the α1141–1160 peptide may be expected to disrupt this interaction, thus displacing the 32P-β from core polymerase, and the results showed that this is in fact the case (Figure 2C, lane 6). To demonstrate specificity of this interaction further, heterologous clamps were tested for displacement of the Pol III core·32P-β interaction. The addition of excess yeast PCNA clamp or phage T4 gp45 clamp did not displace core from 32P-β (Figure 1C, lanes 4 and 5, respectively). Nor did the α-peptide bind to human 32P-PCNA or inhibit the binding of 32P-PCNA to the cell-cycle regulator p21WAFI (data not shown). As expected from their ability to compete with DNA polymerase for β, the β-binding peptides α1140–1159 and α1141–1160 were inhibitory to Pol III core when added to a replication reaction using primed M13mp18 ssDNA as template; core requires β for synthesis of this substrate (Figure 1D). Figure 2.Alanine scan analysis of α C-terminal peptide binding to β. (A) Peptides were pre-bound to streptavidin-coated microtiter plates and probed with 32P-β as in Figure 1A. A clear spot indicates a critical residue for 32P-β binding. (B) Peptides were used to compete Pol III core off the 32P-β·core complex in the native PAGE mobility shift assay, as in Figure 1C. (C) Purified δ protein (20 pmol) was fixed to microtiter plates and probed with 32P-β in the presence of the indicated peptide. Retention of 32P-β in the well indicates that a critical residue has been changed to Ala. Download figure Download PowerPoint Similar binding assays as described above were performed on peptides spanning the N-terminal region of α and the region of the protein from residue 812 to 991 (gray boxes in the key of Figure 1A), with negative results (data not shown). Since the interaction could require a specific conformation not provided by 20-mer peptides, our results do not exclude the possibility that additional sites of contact between α and the β-clamp could be present in this area, as suggested by the studies of Kim and McHenry (1996). On the other hand, our results do not detect the interaction observed by Dalrymple et al. (2001), in which a sequence corresponding to region 918–926 of α (sequence IGQADMFGV), fused onto a reporter protein, resulted in an interaction with the β-clamp using a yeast two-hybrid assay. Peptides spanning this region do not show binding to the β-clamp using the assays and conditions described here (data not shown). Alanine-scan analysis of the α C-terminal peptide To identify residues at the C-terminus of the α-subunit of Pol III that are critical for interaction with β, we performed an alanine scan analysis of the α1141–1160 peptide. These peptides were used in three different assays: (i) direct binding of 32P-β to biotinylated peptide immobilized in wells of a microtiter plate (Figure 2A); (ii) ability to displace Pol III core from a 32P-β·core complex observed in a native polyacrylamide gel (Figure 2B); and (iii) ability to displace 32P-β from δ-subunit immobilized in wells of a microtiter plate (Figure 2C). The δ-β displacement assay is based on the previous finding that binding of α and δ to β is sterically exclusive, and that the same single amino-acid changes on β that abolish interaction with α also abolish interaction with δ, suggesting that α and δ share a common site on β (Naktinis et al., 1996). This conclusion is supported here by the finding that peptide α1141–1160 competes 32P-β off the δ subunit (Figure 2C). While most alanine mutants of the α1141–1160 peptide retain full activity for binding 32P-β, mutation of residues Q1154, L1157 and F1159 to alanine clearly diminished the activity of the peptides in all three assays. Interestingly, peptide D1160A showed no retention of 32P-β on the microtiter plate (Figure 3A), but was almost as effective as wild-type peptide in competition assays (Figure 3B and C). Since deletion of residue D1160 (peptide α 1140–1159) in the experiment shown in Figure 1A did not impair binding to 32P-β, we suggest that the presence of alanine at the position of D1160 lowers the affinity of peptide for the β-clamp. The large concentration of peptide used in the competition assays of Figure 1B and C may overcome the deficiency. Figure 3.An α-subunit truncated in the C-terminal seven residues loses affinity and function with the β-clamp. (A) Fluorescently labeled β-clamp (βOG) at residue 333 was used in KD measurements with wild-type α (squares) and αΔC7 (circles). (B) KD measurements were obtained over a 0–230 mM range of NaCl. (C) Wild-type core (squares) and core reconstituted using αΔC7 (circles) were assayed for processive DNA synthesis on singly primed M13mp18 ssDNA in the presence of the β-clamp and γ-complex. Download figure Download PowerPoint Deletion of the C-terminal seven residues of α greatly reduces interaction with the β-clamp In light of the experiments described above, we deleted the seven C-terminal residues of α that contain the three critical β-interactive residues identified in the alanine scanning experiments. This C-terminal deletion will be referred to here as αΔ7. We then labeled the β-clamp with the fluorescent 488 Oregon Green (OG) probe at the single exposed Cys333, and used fluorescence to determine the affinity of the labeled β-clamp for wild-type α and for αΔC7. Cys333 is on the opposite face of β from where α binds, and OG-labeled βOG was as active as wild-type β in DNA synthesis activity assays with γ-complex and core (data not shown). Titration of α and αΔ7 into βOG reveals that αΔC7 has a 10-fold reduced affinity for the β-clamp (Figure 3A; KD of 1.16 μM versus 0.11 μM at 100 mM NaCl). The residual binding of αΔC7 for the β-clamp could be due to additional interaction points between the two proteins, perhaps in regions distinct from the C-terminal region. Alternatively, C-terminal residues immediately upstream could participate in binding β via protein backbone contacts with the clamp, as in the p21·PCNA complex (Gulbis et al., 1996). This possibility is supported by our earlier study of αΔ20 (deletion of the C-terminal 20 residues), which binds β 100-fold weaker than wild-type α (López de Saro et al., 2003). In this case, the upstream residues that contribute peptide backbone contacts to β would not be detected by the alanine scanning experiments described above. This residual affinity of αΔC7 for the β-clamp was clearly salt dependent (Figure 3B), suggesting that the contribution of the last seven residues has a strong hydrophobic component, consistent with the requirement of L1157 and F1159 in the interaction of α with β. DNA synthesis assays performed with αΔC7 reconstituted with the ϵ and θ subunits to form Pol III (αΔ7) core showed that its activity in a β-clamp-dependent replication assay is considerably lower than that of wild-type core (Figure 3C). This result supports the conclusion that the extreme C-terminal seven residues of α are important to functional interaction with β. Both core complexes displayed similar activities in a β-independent extension assay using gapped DNA as a substrate (not shown), indicating that αΔC7 is not impaired in its catalytic activity. Pol III core interacts with β at the same locus as the δ subunit of the clamp loader During lagging strand synthesis, the clamp loader (γ-complex) loads β-clamps onto multiple RNA primers. The KD of γ-complex for β in solution is 8 nM, ∼30-fold lower than that of the Pol III core·β complex (250 nM). However, when β is on DNA this affinity is reversed, resulting in a KD value for the Pol III core·β of <5 nM (Naktinis et al., 1996) and a reduced affinity of the γ-complex for β (Turner et al., 1999; Ason et al., 2000). This DNA-modulated competition between clamp loader and polymerase for β generates a molecular switch that assures an ordered sequence of events during the assembly of the replicase at a primed site (Ason et al., 2003). A previous study on the site of interaction of δ subunit (of γ-complex) with β, and α subunit (of Pol III core) with β, demonstrated that both proteins interact with the clamp near the C-terminus, which is near the hydrophobic pocket between β domains II and III (Naktinis et al., 1996). Here, as shown in Figure 2C, we find that the 20-mer peptide derived from the C-terminus of α displaces δ from 32P-β. This result indicated that the α and δ proteins bind the same site on β, rather than competing by steric occlusion, in which two large proteins bind different sites on β, but are too close for both α and δ to bind β at the same time. The ability of the α C-terminal peptide to compete δ from β suggests that this peptide may inhibit the clamp loading reaction. Indeed, the peptide inhibits loading of the β-clamp on DNA by γ-complex, but a related peptide mutated in a single residue (Q1154A) does not (Figure 4). These results further support the conclusion that the site of interaction of δ and α with β is the same, namely the hydrophobic pocket between domains II and III of the β ring, which is near the β C-terminus. Figure 4.The α C-terminal peptide inhibits β-clamp loading by the γ-complex. (A) In the presence of ATP, the γ-complex (γ3δδ′χψ) loads 32P-β onto primed M13mp18 ssDNA, resulting in a complex with a molecular mass that elutes earlier than free 32P-β in gel filtration. (B) The plot shows the gel filtration profiles of reactions without peptide added (squares), or with a peptide that does not interact with 32P-β (Q1154A) (circles), or with wild-type α1141–1160 peptide that binds and inhibits the clamp loading reaction (diamonds). Download figure Download PowerPoint Conserved residues at the C-terminus of Pol II, Pol III and Pol IV interact at the same site of β The α C-terminal residues identified by alanine scanning as being essential for interaction with β appear to be present in the C-terminal sequences of Pol II and Pol IV, although the spacing is somewhat different (Figure 5A). A peptide corresponding to the C-terminus of Pol IV has been shown recently to interact with β directly, and a C-terminal deletion of this Y-family polymerase no longer functions with β (Lenne-Samuel et al., 2002). The experiment shown in Figure 5B (upper panel) demonstrates that immobilized peptides derived from the C-terminus of either Pol II or Pol IV interact with 32P-β and retain 32P-β in ELISA plates. If these Pol II and Pol IV peptides bind β at the same site, they should be capable of competing 32P-β off δ protein. The experiment shown in Figure 5B (lower panel) illustrates that this prediction is valid. Immobilized δ retains 32P-β in the well of a microtiter plate, and the addition of C-terminal peptide derived from α, Pol II or Pol IV displaces 32P-β from the immobilized δ. Further, the Pol II and Pol IV proteins also displace Pol III core from the Pol III core·β complex in the native PAGE assay, as shown in Figure 5C. The 20-mer peptides derived from the C-terminus of Pol II and Pol IV also inhibit β-dependent replication by Pol III core (data not shown). Figure 5.Pol II and Pol IV interact with the β-clamp via extreme C-terminal sequences. (A) C-terminal sequences from Pol III, Pol II and Pol IV. Conserved residues that correspond to residues in E.coli α needed to bind β highlighted. (B) Synthetic biotinylated peptides derived from the C-terminus E.coli Pol II and Pol IV bind 32P-β as determined using streptavidin-coated microtiter plates (upper panel), and by ability to disrupt the δ·32P-β complex (lower panel). (C) C-terminal peptides from Pol II and Pol IV displace Pol III core from 32P-β·core complex in a native PAGE assay (as in Figure 1C). (D) The native PAGE assay was used to challenge the 32P-β·core complex with C-terminal Pol II peptide (Pol II764–783) (left panel) or the 32P-β·Pol II complex with a C-terminal α peptide (α1141–1160) (right panel). Peptide was added in reactions 3–10 as follows: 500, 500, 250, 125, 62.5, 31.2, 15.6 and 7.8 μM, respectively. Concentrations of Pol III core (left panel) and Pol II (right panel) were 500 nM and 925 nM, respectively. (E) Fluorescence KD measurements of the interaction between Pol III core (upper left panel) or Pol II to βOG as described in the Materials and methods. The lower panels show the interaction of wild-type β with rhodamine-labeled peptides corresponding to the C-terminal 20 residues of α (left panel) and Pol II (right). Download figure Download PowerPoint The alignment of the C-terminal tails of various polymerases reveals slight differences in the presence and/or spacing of the residues that are important for α to bind β (see Figure 5A). To assess the importance of these variations to the relative affinity to β, we compared the Pol II and Pol III C-terminal peptides for their effectiveness in disrupting polymerase·clamp complexes. In Figure 5D (upper panel) we titrated the 20-mer peptide of Pol II into the 32P-β·Pol III core complex, and in the lower panel, the C-terminal Pol III α 20-mer peptide was titrated into 32P-β·Pol II complex. The results demonstrate that the peptides have similar efficiencies in disrupting the polymerase-clamp complex. We next determined the apparent KD of Pol III core·β (51 ± 9 nM) and Pol II·β (120 ± 23 nM) using βOG (Figure 5E, upper panels). To compare the relative affinity of the C-terminal 20 residues of both polymerases for β, we used synthetic peptides labeled at their N-terminus with rhodamine (TAMRA); the apparent KD values calculated from titration measurements were 3.2 μM for the α peptide and 3.5 μM for the Pol II peptide, revealing that despite the differences in sequence, both peptides bind to the clamp with very similar affinities (Figure 5E, lower panels). Calculation of the free energy of the polymerase·β interaction indicates that ∼75% of the interaction energy between Pol III and β resides in the C-terminal 20 residues (ΔG = 9.94 kcal/mol for Pol III·β, ΔG = 7.50 kcal/mol for Pol III peptide·β) as does 79% of the interaction energy between Pol II and β (ΔG = 9.45 kcal/mol for Pol II·β, ΔG = 7.44 kcal/mol for Pol II peptide·β). DNA polymerases I and V (UmuC) also interact with β in the same locus as Pol III Our previous studies have shown that the β-clamp can stimulate the Klenow fragment of DNA polymerase I in synthesis on SSB-coated, singly primed M13mp18 ssDNA (López de Saro and O'Donnell, 2001). However, the C-terminus of the Klenow fragment lacks a sequence containing the β-binding residues of the Pol III α-subunit. Consistent with this, we detected no binding to the β-clamp of a C-terminal 20-residue peptide derived from the Klenow sequence (data not shown). However, the Pol III α subunit peptide (α 1141–1160) did inhibit β-stimulated DNA synthesis by Pol I Klenow fragment (Figure 6A). In this experiment the β-clamp was loaded onto the primed DNA before the peptide and Pol I (Klenow fragment) were added. Hence, the observed inhibition is likely the result of disruption of the Pol I·β contact by the peptide. Further, 32P-β is retained in wells by immobilized Klenow fragment (Figure 6B), suggesting that the site on β that is bound by the α C-terminal peptide and the Pol I Klenow fragment is the same, and that both polymerases interact with the clamp in a competitive manner. Figure 6.Pol I (Klenow fragment) and Pol V also bind the β-clamp in the same locus as δ, Pol III, Pol II and Pol IV. (A) Stimulation of DNA synthesis by th