View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 82, 535-542, August 25, 1995, Copyright 0 1995 by Cell Press Prokaryotic Meeting Review and Eukaryotic Replicons Joel A. Huberman complexed with viral DNA polymerase molecules, are po- Department of Molecular and Cellular Biology sitioned at the 3’ ends of the parental duplex. Using the Roswell Park Cancer Institute penultimate 3’ deoxyribosylthymine (dT) as template, the Buffalo, New York 14263 polymerase then catalyzes the formation of a covalent bond between deoxyribosyladenine (dA) and a serine of TP. Next, the TP-dA slides back 1 nt to position the dA adjacent to the 3’ terminal dT of each strand. The poly- The replicon model, proposed by Jacob and Brenner merase then catalyzes processive elongation of the new (1963) to explain the regulation of prokaryotic DNA replica- protein-primed strands, using the 3’-ended strands as tion, has proved remarkably robust. Despite the manyvari- templates and displacing the 5’-ended strands. This “slid- ations in initiation mechanism discovered since 1963, the ing-back” initiation mechanism, which provides an oppor- replicon model may be applicable, with minor modifica- tunity during subsequent replication rounds to correct nu- tions, to regulation of DNA replication in all organisms. cleotides incorrectly incorporated at the first step, appears For several years, the Replicon Club of Paris, a group to be a common feature of linear replicons employing the of French scientists interested in regulation of replication, TP mechanism. has been meeting to discuss new results. This year, with Rolling Circle support from the French Centre National de la Recherche S. D. Ehrlich (Institut National de la Recherche Agrono- Scientifique (CNRS), the Replicon Club sponsored an in- mique [INRA], Jouy en Josas, France) discussed the Rep ternational Jacques Monod Conference on Prokaryotic protein of pC194, a rolling circle plasmid of gram-positive and Eukaryotic Replicons. The meeting was organized by bacteria. A major difference between pC194 and the well- G. Buttin (Institut Pasteur, Paris) with assistance from A. studied rolling circle replicon @X174 is that @X174 is a Falaschi (International Centre for Genetic Engineering virus with runaway replication, while pC194 is a plasmid and Biotechnology, Trieste, Italy) and M. Kohiyama (Insti- with regulated replication. Thedifferencecan beexplained tut Jacques Monod, Paris) and was held at the CNRS’s by comparison of the two Rep proteins. Both proteins initi- Centre Paul Langevin in the Alpine village of Aussois, ate replication by making nicks at specific sites in one of France, from June 18-22, 1995. There, invigorated by the DNA strands, yielding 3’ ends that serve as primers beautiful surroundings and excellent cuisine, scientists and S’ends covalently attached to a Rep protein tyrosine. from around the world provided evidence both for the vital- After one round of synthesis, second nicks are made at ity of the replicon model and for substantial advances in the same sites. The @X174 Rep protein employs a second our understanding of the regulation of replication. tyrosine to catalyze the second nick, permitting retention of the5’end and, ultimately, reinitiation. In contrast, PC194 Variety of Replicons appears to use glutamic acid-catalyzed hydrolysis for the The original replicon model (Figure 1) suggested that repli- second nick, thus losing bond energy and preventing reini- cation is positively controlled by an initiator protein that tiation. acts on a single replicator to initiate replication of a circular Rolling circle replication is also employed by geminivi- replicon. Extension of the model to other organisms re- ruses, whose small circular single-stranded genomes rep- quires allowance for multiple linear chromosomes in eu- licate via double-stranded intermediates in the nuclei of karyotic cells, each possibly having multiple replicators plant cells. Since replication is catalyzed entirely by en- (Figure 1). This extension requires that replicon be rede- zymes from the host cell (except the viral Rep protein) and fined to mean the stretch of DNA replicated from a single viral DNA is packed into chromatin, study of geminivirus replicator. An updated model also requires allowance for replication appears to be a promising way to learn more the possible existence of multiple initiator proteins, which about DNA replication and the cell cycle in plants. B. Gro- may form a complex (as in Figure 1) or may separately nenborn (Institut des Sciences V&g&ales, Gif sur Yvette, bind to different portions of the replicator, and recognition France) reported that geminivirus Rep proteins nick the that interactions between initiators and replicators are typi- origin via a tyrosine and have a DNA-independent ATPasel cal binding reactions, governed by the laws of mass action GTPase activity that is essential for in vivo replication and (Figure 1). Consequently, if the concentration of initiators reminiscent of the GTPase in signal transduction G pro- is high or their specificity is low, most or all DNA sequences teins. C. Gutierrez (Centro de Biologia Molecular Sever0 may be able to serve as replicators. Ochoa, Madrid) showed that geminivirus Rep contains an At the meeting in Aussois, developments relating to a LXCXE motif capable of mediating stable binding to the wide variety of replicons were presented. human retinoblastoma (Rb) protein. This motif is essential Protein Priming for in vivo replication, suggesting that viral replication is The Bacillus subtilis phage, @29, has a linear double- linked to cell cycle control and that plants, like animals, stranded DNA with a viral terminal protein (TP) covalently may contain Rb homologs that inhibit entry into S phase. attached to its 5’ ends. L. Blanc0 (Centro de Biologia Mo- Unidirectional 8 lecular Sever0 Ochoa, Madrid) described the mechanism C. Bruand and E. Le Chatelier (INRA, Jouy en Josas, of initiation of @29 replication. For each round, new TPs, France) described the pAMP1 replicon, a plasmid of gram- Cell 536 hr after infection. Evidence for interactions between UL9 and cellular DNA polymerase a, but not the viral DNA polymerase, suggests that initiation at viral origins may be accomplished by UL9 and cellular polymerases. Later Original Model rolling circle replication is independent of UL9 but requires the other viral replication proteins, which, according to physical and electron microscopic evidence, may exist as a complex. Gel shift and footprinting experiments described by P. Elias (Gdteborg University, Sweden) suggest that a protein complex may also function at the viral origin. The complex would consist of two dimers of UL9, interacting through their C-terminal domains both with specific binding sites in the origin and with four viral single-stranded DNA-binding proteins, which are called ICP-8. Since ICP-8 binds more Replicator Initiator Replicator Replicator Initiator tightly to single-stranded DNA than to UL9, it is likely that Gene1 Gene2 once single-stranded DNA is exposed at the origin by un- Figure 1. Past and Present Replicon Models winding of parental strands, ICP-8 is transferred to the (Top) Model proposed by Jacob and Brenner (1963). (Bottom) Model single-stranded DNA. arising from discussions at the Aussois meeting described in this re- During the early stage of bacteriophage h replication, view. initiation is mediated by the phage-encoded 0 protein at orik. Under in vivo conditions, transcription near orid, usu- ally from the PR promoter, is also required to initiate replica- positive bacteria, which bears some resemblance to the tion. G. Wegrzyn (University of Gdansk, Poland)discussed well-known ColEl replicon. For pAM81, a plasmid-encoded circular molecules derived from h but missing most of the positive regulator, the RepE gene product, is essential for hgenome except the origin region. He presented evidence replication, and DNA polymerase I is replaced by DNA that 0 protein, along with other proteins involved in initia- polymerase Ill holoenzyme (Hpollll) about 200 bp down- tion, is retained at one of the two daughter origins during stream of ori. According to M.-A. Petit and L. Janniere each replication round. Such inherited replication com- (INRA, Jouy en Josas, France), this polymerase switch is plexes may mediate further replication rounds. Runaway aided by the pAM81 resolvase, which binds tightly about replication is prevented by the requirement that each 250 bp downstream of ori, thereby blocking polymerase round be activated by transcription at PR. Such transcrip- I and creating a D loop with an exposed primosome assem- tion is mediated by the host dnaA protein, which is present bly site where an Hpollll-based replication fork can effi- in high concentration during only a limited portion of each ciently be set up. V. Bidnenko (INRA, Jouy en Josas, cell cycle. France) showed that the plasmid-encoded topoisomerase, Bidirectional 8 topp, also assists by relaxing plasmid DNA and thereby The single initiator protein of Escherichia coli, encoded removing thedriving force for strand unwinding when poly- by the dnaA gene, governs initiation at the single repli- merase I reaches about 190 bp downstream of ori. The cator, oriC (reviewed by Kornberg and Baker, 1991). Inter- presenters proposed that similar mechanisms may facil- actions between the dnaA protein and oriC were described itate polymerase switching in ColEl and eukaryotic rep- by W. Messer (Max-Planck-lnstitutfur Molekulare Genetik, I/cons. Berlin). Within 0% there are four 9 nt repeats, called dnaA Previously studied prokaryotic and eukaryotic circular boxes, that serve as specific binding sites for the dnaA plasmids containing two replication origins utilize only one protein. Measurements of binding to the individual boxes origin at a time. J. B. Schvartzman (Centro de Investigaci- and tocomplete o&revealed that binding is highly context ones Biologicas, Madrid) reported that occasionally mole- dependent. The dnaA protein does not contain any of the cules of the plasmid pPl21, containing two unidirectional known DNA-binding motifs.