Proliferating cell nuclear antigen (PCNA) is an essen- tial protein found in all proliferating eukaryotic cells, and carries out crucial roles in both DNA replication and DNA repair. First described as an autoantigen 1 detectable in the nuclei of proliferating cells, it was subsequently identified as an S-phase protein and dubbed 'cyclin'2. This potentially confusing name was gradually phased out as the importance of the cyclins and cyclin-dependent kinases (CDKs) became apparent. PCNA carries out crucial roles in three major forms of eukaryotic DNA repair and is central to leading, and probably lagging, strand DNA repli- cation. How one protein can carry out such a range of different and diverse roles has been the subject of intensive research over the past few years. It now ap- pears that control of PCNA activity depends to some extent on the nuclear environment, determined by regulatory proteins that are induced in response to changes in growth conditions or following DNA damage. Discoveries over the past ten years of an extensive network of proteins that can bind to PCNA support the idea that some or all of them might modulate PCNA activity. The possibility that com- petition between these proteins for association with PCNA might regulate DNA replication and repair is the main theme of this review. PCNA gene and protein structure Consistent with its essential function in all eu- karyotes, PCNA has been highly conserved through evolution of animals and plants at both the protein and the DNA sequence level 3. The cDNA open read- ing frame of human PCNA predicts an acidic 261 amino acid protein of 29 kDa (Ref. 4), although the apparent molecular mass on SDS-PAGE is 36 kDa. This might well reflect structural flexibility in the region of residues 128-150 rather than posttrans- lational modification s. Crystallography studies 6,7 have shown that PCNA can self-associate as a trimer, forming a hexagonal ring with sixfold pseudosymmetry and a central hole - hence the common 'doughnut' description (Fig. 1). Each PCNA monomer comprises 18 ~ sheets and four c~ helices, which form two structurally similar do- mains. In the centre of the trimer is a cavity that ap- pears to be large enough to accommodate duplex DNA. This cavity is lined with positively charged ~ helices, consistent with interaction with the negatively charged sugar-phosphate backbone of DNA. The ring-shaped PCNA molecules are structurally similar to the replication-associated dimeric 13 clamp of Escherichia coli DNA polymerase (pol) III holoenzyme, despite sharing only -5% identity of sequence 6-8 - a remark- able example of structure-function conservation through evolution. It has been suggested that PCNA slides along duplex DNA in a manner similar to that of the bacterial protein 9. Recent neutron-scattering experiments strongly sug- gest that all recombinant human PCNA is trimeric in solution (U. Hubscher, pers. commun.). The func- tional importance of self-association is demonstrated by a single point mutation at the monomer interface that disrupts PCNA trimerization and concomitantly abolishes its replication activity1°. A longer, 365 amino Who binds wins: competition for PCNA rings out cell-cycle changes PCNA, proliferating cell nuclear antigen, is a pivotal protein in DNA replication, DNA repair and possibly cell-cycle control. The protein has a trimeric ring structure that might slide along duplex DNA and form a platform for association with a variety of proteins, in particular holding the DNA polymerases in close association with their template. This article reviews evidence suggesting that the activity of PCNA in replication and repair is coordinated within the cell cycle by cooperative and competitive interactions with an extensive network of enzymes and regulat~oryproteins. acid, embryonic form of eukaryotic PCNA has been identified in carrot (Daucus carota)11 that might be able to form dimers, also -90 kDa. Although it is not yet clear whether only trimers or different multimers exist in mammalian cells, it is possible that regu- lated association between PCNA monomers might be important in determining function. The toroidal structure of PCNA revealed by X-ray crystallography6,7 has distinct front and back faces that might provide a variety of sites for interaction with other proteins. This asymmetry could also de- fine the directionality of PCNA movement as it slides along DNA. Additionally, the 'loop' region between the twin domains of each monomer is a highly im- munogenic exposed site lz that appears to be impor- tant for interaction with other proteins 7A3A4, and the structure shown in Figure I clearly demonstrates the potential of PCNA to act as a 'platform' for such protein-protein interactions. The functional partners of PCNA are becoming increasingly recognized as crucial in regulating its roles in replication and repair. PCNA in DNA replication The subcellular distribution of PCNA as detected by immunofluorescence after methanol fixation varies through the cell cycle in a manner consistent with a role in nuclear DNA replication ls,16. Studies with both synchronized and asynchronous cell populations show that PCNA is localized to the nucleus during G1 of the eukaryotic cell cycle (Fig. 2a). It then accumulates in larger subnuclear 'clumps' in S phase that represent matrix-associated replication factories (Fig. 2b). This subpopulation of total nuclear PCNA Lynne Cox is in the Deptof Biochemistry, University of Oxford, Oxford, UK OX1 3QU. E-mail: Iscox@ bioch.ox.ac.uk 493 trends in CELL BIOLOGY (Vol. 7) December 1997 Copyright © 1997 ElsevierScience Ltd. All rights reserved. 0962-8924/97/$17.00 PIl: s0962-8924(97)01170-7