Clamp loading, unloading and intrinsic stability of the PCNA, ¯ and gp45 sliding clamps of human, E. coli and T4 replicases Nina Yao1, Jennifer Turner1, Zvi Kelman1,a, P. Todd Stukenberg2,b Frank Dean1, David Shechter1, Zhen-Qiang Pan2, Jerard Hurwitz2 and Mike O’Donnell1, * 1Microbiology Department and Hearst Research Foundation, *Howard Hughes Medical Institute, Cornell University Medical College, 1300 York Avenue, New York, HY 10021, USA, and 2Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Centre, 1275 York Avenue, New York, NY 10021, USA Communicated by: Martin Gellert Abstract Background: The high speed and processivity of oligomeric state. The T4 gp45 clamp is a much replicative DNA polymerases reside in a processivity less stable trimer than PCNA (Kd % 250 nM) and factor which has been shown to be a ring-shaped requires association with the polymerase to stabilize protein. This protein (‘sliding clamp’) encircles it on DNA as observed previously. The consequence DNA and tethers the catalytic unit to the template. of this cooperation between clamp and polymerase Although in eukaryotic, prokaryotic and bacter- is that upon finishing a template and dissociation of iophage-T4 systems, the processivity factors are the polymerase from DNA, the gp45 clamp sponta- ring-shaped, they assume different oligomeric neously dissociates from DNA without assistance. ¯ states. The Escherichia coli clamp (the ¯ subunit) is However, the greater stability of the PCNA and active as a dimer while the eukaryotic and T4 phage clamps on DNA necessitates an active process for clamps (PCNA and gp45, respectively) are active as their removal. The clamp loaders (RF-C and ° trimers. The clamp can not assemble itself on DNA. complex) were also capable of unloading their Instead, a protein complex known as a clamp loader respective clamps from DNA in the presence of ATP. utilizes ATP to assemble the ring around the Conclusions: The stability of the different clamps in primer-template. This study compares properties solution correlates with their stability on DNA. of the human PCNA clamp with those of E. coli and Thus, the low stability of the T4 clamp explains the T4 phage. inability to isolate gp45 on DNA. The stability of the Results: The PCNA ring is a stable trimer down to a PCNA and ¯ clamps predicts they will require an concentration below 100 nM (Kd % 21 nM). On DNA, unloading factor to recycle them on and off DNA the PCNA clamp slides freely and dissociates from during replication. The clamp loaders of PCNA and ¯ ¯ DNA slowly (t1/2 % 24 min). is more stable in double as clamp unloaders presumably for the solution (Kd <60pM) and on DNA (t1/2 % 1 h) than purpose of clamp recycling. PCNA which may be explained by its simpler Introduction * Corresponding author: Fax: +1 212 746 8587. The sliding clamps of chromosomal replicases are ring- a Present address: Department of Molecular Biology and Genetics, shaped proteins that encircle DNA and tether the John Hopkins University, School of Medicine, 725 N. Wolfe replicase to the template for highly processive chain Street, Baltimore, MD 21205-2195, USA. elongation (reviewed in Kuriyan & O’Donnell 1993). b Present address: Department of Cell Biology, Harvard Medical The clamp loader recognizes a primed template School, Boston, MA 02115, USA junction and couples ATP hydrolysis to assemble the 5 Blackwell Science Limited Genes to Cells (1996) 1, 101–113 101 N. Yao et al. clamp around DNA. The eukaryotic clamp is the similar to in its stability on DNA and requirement for a proliferating cell nuclear antigen (PCNA), the pro- clamp unloader for its removal. karyotic clamp is the subunit and the T4 phage clamp is the product of gene 45 (gp45) (reviewed in Kuriyan Results & O’Donnell 1993). The E. coli sliding clamp is a dimer (Stukenberg et al. 1991; Kong et al. 1992) while To directly follow the properties of PCNA, and gp45 the eukaryotic and T4 phage sliding clamps are formed in these studies we have radiolabelled them. All three by trimers (Krishna et al. 1994; Jarvis et al. 1989). In each were tritiated by reductive methylation, a modification case, the clamps have been shown to slide on that results in a 3H-methyl group on one or two lysine DNA freely (Stukenberg et al. 1991; Gogol et al. residues of each molecule (the charge is retained at 1992; Burgers & Yoder 1993; Tinker et al. 1994). The physiological pH) (Kelman et al. 1995a). Additionally, clamp loaders in each of these systems are composed some studies were performed using 32P-labelled clamps of multiple subunits. The E. coli clamp loader is the using and PCNA containing a kinase recognition five-subunit complex, the eukaryotic clamp loader is motif on the C- or N-terminus, respectively. the five protein RF-C (also called activator-1) and the T4 clamp loader is the gene protein 44/62 complex In solution, the trimer clamps are less stable (also a five subunit structure, reviewed in Kelman & oligomers than the dimer clamp O’Donnell 1994). A major difference among these systems is that the and PCNA clamps can be Previous studies have shown that upon transfer of sliding isolated on DNA by gel filtration whereas the T4 clamps to DNA by their respective clamp loaders, the gp45 clamp requires association with its DNA PCNA and clamps can be isolated on DNA by gel polymerase, the product of gene 43 (gp43), to be filtration, but the T4 gp45 clamp can not (Wickner isolated on DNA. 1976; O’Donnell 1987; Maki & Kornberg 1988; Lee & In E. coli, the complex acts catalytically to assemble Hurwitz 1990; Burgers 1991; Capson et al. 1991; the clamp on DNA and can be removed from the Richardson et al. 1991). One possible explanation for clamp-DNA complex (Wickner 1976; Maki & Korn- the difference (in ability to form stable clamps on DNA) berg 1988; Stukenberg et al. 1991). The DNA amongst the three systems could be the inherent stability polymerase III (Pol III core) then associates with the of the oligomeric structure of the clamps themselves. In clamp on DNA for processivity. However, an additional Fig. 1 the oligomeric structure of these clamps were ‘connector’ protein called ( binds two molecules of Pol examined by gel filtering a mixture of all three in III core and one complex to produce a tightly solution. They were analysed at a high concentration associated particle called Pol III* (Onrust et al. 1995). (2.5 "M, Fig. 1A) and then at a 50-fold lower concen- The two Pol III core polymerases are thought to tration (50 nM, Fig. 1B). To follow these proteins we replicate the leading and lagging strands concurrently added a small amount of 3H-protein and analysed their (Sinha 1980; Kornberg & Baker 1992). elution patterns by fluorography after SDS-polyacryl- In eukaryotes and T4 phage, no such particle amide gel electrophoresis. At 2.5 "M, each clamp eluted containing both polymerase and clamp loader has at a position consistent with its native oligomeric state: been identified. Although it is presumed that the PCNA as a trimer (86.7 kDa, fractions 28–31), as a clamp loader and polymerase assemble together with dimer (81.2 kDa, fraction 31–34) and gp45 as a trimer the clamp on the DNA in these systems, it remains (74.1 kDa, fractions 31–34). However, at 50 nM, the possible that the clamp loader acts catalytically resulting oligomeric states of the trimeric clamps begin to change, in only the clamp-polymerase complex, as observed in but remained a stable dimer. At 50 nM, PCNA showed the E. coli system. Indeed, the catalytic action of the T4 two distinct peaks; one correlated with its trimeric form, gp44/62 complex has been documented (Kaboord and the other was a smaller species that eluted in & Benkovic 1995). In eukaryotes, both DNA poly- fractions 37-40. At this low concentration, gp45 eluted 32 merase (pol ) and DNA polymerase (pol ) interact entirely as a smaller species in fractions 37–43. P- of with the PCNA clamp (Burgers 1991; Lee et al. 1991a). high specific activity was used to examine the behaviour 32 In this report the inherent strength of the oligomeric of at even more dilute conditions. P- remained a structure of the clamps in solution, and their stability on stable dimer even upon dilution to 67 pM and incubation DNA have been examined. Despite the apparent at 37 VC with 1 M NaCl in buffer D for 6 h (J. similarities of PCNA and T4 gp45 (both are trimers Andjelkovic & M. O’Donnell, unpublished data). The and less stable oligomers than the dimer), PCNA is possibility that the monomer elutes at the same 102 Genes to Cells (1996) 1, 101–113 5 Blackwell Science Limited Comparison of DNA sliding clamps the dimer, as expected, supporting the conclusion that the wild type dimer remains a stabile oligomer at the lowest concentrations tested. Ring shaped trimer oligomers are expected to be less stabile oligomers that ring shaped dimers (Kelman et al. 1995b). In a protein ring each protomer has two contacts, one with each adjacent protomer. In the case of the trimer, upon dissociation of one protomer, the remaining two protomers now have only one contact, which should lead to cooperative disassembly of the entire ring. Thus, it seems reasonable to expect that the smaller species observed upon dilution of PCNA is the monomer. Further dilution did not result in a third, smaller species, as would be expected if PCNA disassembled in stages from trimer-to-dimer- to-monomer. A recent study has shown that the PCNA trimer dissociates into a putative dimer form upon dilution (Zhang et al.