Embryonic PCNA: a missing link?

PCNA, the processivity factor of the eukaryotic replicative DNA polymerase [1], is the functional homolpgue of the [3 subunit of Escherichia coli DNA polymerase III holo-enzyme [2,3]. Biochemical studies have shown that the E. coli 3 subunit acts as a sliding clamp on duplex DNA; it has a ring shape that encircles DNA and it binds directly to Pol III, tethering it to DNA to achieve highly processive synthesis [4]. The crystal structure of the 13 subunit indeed showed it to be a dimeric closed ring with a central cavity large enough to encircle DNA [5]. Each monomer is composed of three globular domains, each with the same chain-folding pattern and very nearly the same three-dimensional structure, so the dimer has a six-fold symmetry (Fig. 1). The PCNA amino-acid sequence is only two-thirds the length of that of the 13 subunit, so PCNA was thought to have only two domains per monomer and to trimerize to form a six-domain ring [5]. Biochemical data have been obtained that demonstrate PCNA to be a sliding clamp (like the 13 subunit), and the crystal structure of Saccharomyces cerevisiae PCNA shows it is a six-domain ring, arranged as a trimer as predicted [6]. The structural similarities are striking, as the chain folds of the globular domains are the same within the 13 subunit and PCNA. To determine at which point in evolution the clamp changed from a dimer to a trimer, we searched the databases for 3-subunit and PCNA sequences. The six completely sequenced prokaryotic 13 genes (dnaN) encode similar-sized proteins (365-378 residues), and 12 PCNA genes encode proteins approximately two-thirds this size (256-268 residues). A possible missing link between dimer and trimer was found, in a form of PCNA observed during embryogenesis of Daucus carota, the carrot. Two distinct PCNA genes were isolated from D. carota [7], one encoding a 264-residue polypeptide (29.2 kD), and the other a 365-amino-acid polypeptide (40.1 kD). These PCNAs, like their E. coli and human counterparts, are acidic proteins. The short PCNA is 88 % identical to the amino terminal two-thirds of the long form and shares 65 % identity with human PCNA. The longer PCNA is expressed (along with the short form) during carrot somatic embryo-genesis, when cells proliferate rapidly. This may not be the only example of a long PCNA form in embryogenesis: Leibovici et al. [81 identified a large PCNA transcript during oogenesis of Xenopus …