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PCNA: Structure, Functions and Interactions Oncogene (1997) 14, 629 ± 640 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 REVIEW PCNA: structure, functions and interactions Zvi Kelman Johns Hopkins School of Medicine, Department of Molecular Biology and Genetics, 725 N Wolfe Street, Baltimore, Maryland 21205, USA Proliferating cell nuclear antigen (PCNA) plays an Extensive studies have been conducted on PCNA essential role in nucleic acid metabolism as a component and its functions in the past several years, and PCNA of the replication and repair machinery. This toroidal- has been found to play a critical role in several shaped protein encircles DNA and can slide bi- biological processes that appear disparate but have in directionally along the duplex. One of the well- common a role in DNA metabolism. PCNA is an established functions for PCNA is its role as the essential component of the DNA replication machin- processivity factor for DNA polymerase d and e. PCNA ery, functioning as the accessory protein for DNA tethers the polymerase catalytic unit to the DNA polymerase d (Pold), required for processive chromo- template for rapid and processive DNA synthesis. In somal DNA synthesis, and DNA polymerase e (Pole). the last several years it has become apparent that PCNA PCNA is also required for DNA recombination and interacts with proteins involved in cell-cycle progression repair. In addition, PCNA was shown to interact with which are not a part of the DNA polymerase apparatus. cellular proteins involved in cell cycle regulation and Some of these interactions have a direct eect on DNA check point control. Furthermore, the three-dimen- synthesis while the roles of several other interactions are sional structures of yeast and human PCNA have been not fully understood. This review summarizes the determined and several structure-function relationships structural features of PCNA and describes the diverse have been established. functions played by the protein in DNA replication and The aim of this article is to summarize our current repair as well as its possible role in chromatin assembly understanding of the diverse functions played by and gene transcription. The PCNA interactions with PCNA in DNA metabolism. The association between dierent cellular proteins and the importance of these PCNA and dierent cellular proteins will be described, interactions are also discussed. as well as the roles played by these interactions. Keywords: DNA replication; DNA repair; cell cycle; p53; PCNA; protein-DNA interaction Structure The biochemical data Introduction PCNA is the processivity factor of Pold and thus is the functional homologue of other processivity factors, the b Proliferating cell nuclear antigen (PCNA) was subunit of the Escherichia coli DNA polymerase III identi®ed independently by two dierent groups. holoenzyme (Pol III) and the product of gene-45 of Miyachi et al. (1978) initially identi®ed an auto- bacteriophage-T4. Similarities in the function of these antigen in patients with systemic lupus erythematosis, proteins gave the ®rst indication of the structure of which they named PCNA because the protein was PCNA. observed in the nucleus of dividing cells. At about the PCNA has been described as a `sliding clamp'. The same time Bravo and Celis (1980) used two ®rst evidence for the sliding clamp structure came dimensional gel electrophoresis to identify a protein from the study of the b subunit of E. coli Pol III. In a which was synthesized during the S-phase of the cell series of elegant experiments it was shown that the b cycle which they named cyclin. Subsequent work subunit bound tightly to nicked circular plasmid but demonstrated that PCNA and cyclin are the same readily dissociated upon linearization of the plasmid 29 kDa protein (Mathews et al., 1984). This protein is via sliding over the ends (Stukenberg et al., 1991; Yao now called PCNA; the term cyclin is now used to et al., 1996; reviewed in Kelman and O'Donnell, describe a family of proteins involved in cell cycle 1995b). These results suggested that the b subunit regulation. Genes which encode the PCNA protein binds DNA in a topological fashion, by encircling it. have been isolated from several eukaryotes and The g45 protein has a similar structure. Electron archebacteria and the predicted amino acid sequences microscopy studies showed that g45 protein forms have been found to be highly conserved (Bult et al., structures that resemble `hash-marks' on DNA, and it 1996; Kelman and O'Donnell, 1995a). The chromoso- appears that the DNA is threaded through the protein mal locations of PCNA genes from several species (Gogol et al., 1992). have been determined (e.g. Henderson et al., 1994; Ku Several other lines of biochemical data also supported et al., 1989), as have several PCNA pseudogenes (Ku the idea that PCNA forms a ring around DNA. In the et al., 1989; Yamaguchi et al., 1991). absence of the clamp loader (RF-C) (the complex that assembles the ring around duplex DNA; discussed Received 7 August 1996; revised 15 October 1996; accepted 15 below) it was demonstrated that PCNA can support October 1996 processive replication by Pold on linear DNA with a PCNA: Structure, functions and interactions ZKelman 630 double stranded end, but not on a closed circular very similar to each other and to the E. coli b subunit. template (Burgers and Yoder, 1993). These results The rings are superimposeable; each ring has similar suggested that PCNA could thread itself onto the end dimensions and each has a diameter of the central of the dsDNA molecule and slide along the duplex until cavity large enough to accommodate a dsDNA it reached the 3' end, where it interacted with Pold to molecule (Krishna et al., 1994; reviewed in Kuriyan initiate processive DNA synthesis. Crosslinking experi- and O'Donnell, 1993; Kelman and O'Donnell, 1995a). ments were used in a dierent strategy to demonstrate The PCNA trimer forms a ring with an inner the sliding property of PCNA (Tinker et al., 1994). After diameter of 34AÊ ; this is sucient to accommodate B- assembly around circular plasmid DNA, PCNA could be form DNA (Figure 1). The trimeric ring has sixfold crosslinked to the DNA. Upon linearization of the symmetry as each monomer contains two globular plasmid, however, PCNA could no longer be crosslinked domains. Each domain contains two a helices which to the template, suggesting it has sliding properties line the central cavity and are perpendicular to the (Tinker et al., 1994). Experiments similar to these used to DNA. The a helices are supported by a continuous demonstrate the sliding of the E. coli b subunit were layer of nine b sheet structures all around the outside. performed with human PCNA. This study showed that Surprisingly, although the domains in each monomer PCNA could bind nicked circular plasmid but slid o the do not have sequence similarity they are nearly ends upon linearization (Yao et al., 1996). identical in three-dimensions (Krishna et al., 1994). It is interesting to note that the dimer of the E. coli b subunit is also composed of six domains while each The three dimensional structure monomer contains three globular domains (Kong et The crystal structure analysis of the E. coli b dimer al., 1992), and that several other enzymes involved in showed it to be a ring shaped protein with an inner DNA metabolism share a similar six domain structure diameter sucient to accommodate a dsDNA molecule (Kelman et al., 1995). (Kong et al., 1992). In the paper describing the three In all species studied, PCNA is an acidic protein with dimensional structure of b the authors predicted that low pI (Kelman and O'Donnell, 1995a). The charge PCNA would have a similar structure (Kong et al., distribution on the ring is asymmetric. There is a strong 1992). In fact, the PCNA monomer is only 2/3 the size negative electrostatic potential in the outer surface and a of the b subunit but was shown to form a trimer in net positive electrostatic potential in the central cavity solution (Bauer and Burgers, 1988a; Brand et al., 1994; (Krishna et al., 1994). The negatively charged surface Yao et al., 1996). It was therefore suggested that may prevent nonspeci®c interaction with DNA. Upon PCNA would form a trimeric ring (Kong et al., 1992). assembly around DNA by the clamp loader (see below) Indeed, the overall structures of the human (Gulbis et the positively charged central cavity may stabilize the al., 1996) and yeast (Krishna et al., 1994) PCNAs are interactions between PCNA and DNA. Figure 1 Structure of human PCNA. Left panel: Front view. Twelve a helices line the inner cavity and the outside perimeter is a continuous layer of b sheet structure. The inner and outer diameters of the ring are shown. Right panel: Side view. The width of the ring is shown PCNA: Structure, functions and interactions ZKelman 631 The PCNA trimer is stable both in solution and on are important for rapid chromosomal replication DNA. In solution, the Kd of PCNA dissociation into needed in early embryogenesis (Kelman and O'Don- monomers is 20 nM (Podust et al., 1995; Yao et al., nell, 1995c) and/or for DNA repair (Stillman, 1996). 1996), and the trimer remains stable at 500 mM NaCl. As described above, PCNA exhibits sixfold symme- PCNA is also stable on DNA with an observed half try as a result of having two globular domains in each life of 22 min (Podust et al., 1995; Yao et al., 1996). monomer (Figure 1). Several other proteins which are Several forces may be involved in the stability of the involved in DNA metabolism (e.g. helicase, RuvB) also ring. The X-ray structure revealed that the interfaces have sixfold symmetry and form a hexameric ring between the promoters are formed by parallel b sheets (Kelman et al., 1995).
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