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COMMENTARY Does Transcription by RNA Polymerase Play a Direct Role in the Initiation of Replication?

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COMMENTARY Does Transcription by RNA Polymerase Play a Direct Role in the Initiation of Replication? Journal of Cell Science 107, 1381-1387 (1994) 1381 Printed in Great Britain © The Company of Biologists Limited 1994 COMMENTARY Does transcription by RNA polymerase play a direct role in the initiation of replication? A. Bassim Hassan* and Peter R. Cook† CRC Nuclear Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK *Present address: Addenbrooke’s NHS Trust, Hills Rd, Cambridge CB2 2QQ, UK †Author for correspondence SUMMARY RNA polymerases have been implicated in the initiation of replication in bacteria. The conflicting evidence for a role in initiation in eukaryotes is reviewed. Key words: cell cycle, initiation, origin, replication, transcription PRIMERS AND PRIMASES complex becomes exclusively involved in the synthesis of Okazaki fragments on the lagging strand. A critical step in this DNA polymerases cannot initiate the synthesis of new DNA process is the unwinding of the duplex. chains, they can only elongate pre-existing primers. The opposite polarities of the two strands of the double helix coupled with the 5′r3′ polarity of the polymerase means that RNA POLYMERASES AND THE INITIATION OF replication occurs relatively continuously on one (leading) REPLICATION IN BACTERIA strand and discontinuously on the other (lagging) strand. The continuous strand probably needs to be primed once, usually The first evidence of a role for RNA polymerase came from at an origin. Nature has found many different ways of doing the demonstration that the initiation of replication in this, including the use of RNA primers made by an RNA poly- Escherichia coli was sensitive to rifampicin, an inhibitor of merase (e.g. during replication of the plasmid ColE1 and mito- RNA polymerase, independently of any requirement for chondrial DNA), preformed tRNA (by retroviral reverse tran- protein synthesis (Lark, 1972). Genetic analysis also pointed scriptases), DNA primers generated by endonucleolytic to a direct role (reviewed by Kornberg and Baker, 1992). cleavage (by gpA of øX174) and even a serine OH group (ade- Replication begins at oriC, a ~245 bp region that is highly noviral 55 kDa terminal protein). The discontinuous strand conserved among enteric bacteria; it contains four binding sites must be primed repeatedly to generate short nascent chains for dnaA, the initiator protein, and back-to-back promoters. (Okazaki fragments) and the multiple RNA primers needed are Temperature-sensitive mutations in dnaA are generally lethal invariably synthesized by a special primase, which is an at the non-permissive temperature but can be suppressed by integral part of the replication machinery (see Kornberg and additional mutations elsewhere in the genome. Such suppres- Baker, 1992, for a comprehensive review). This means that sors map to rpoB, the gene encoding the β subunit of RNA RNA synthesis, by RNA polymerases or primases, is polymerase, indicating that altered RNA polymerases can inevitably involved in primer synthesis. This review will con- assist defective dnaA proteins. Some mutations in both the β centrate on the additional roles played by RNA polymerases in and β′ subunits of the polymerase also elevate the copy number initiating DNA synthesis, especially in eukaryotes. Unfortu- of oriC-bearing chromosomes, again implicating this poly- nately no clear view as to their role emerges. merase in the regulation of initiation. The current model for the initiation of replication at origins Fortunately, templates containing oriC can be replicated in unifies the findings from many different organisms (Bramhill vitro and direct experiments show that RNA polymerase is and Kornberg, 1988). A trans-acting initiator protein first binds required, but only under conditions that make unwinding to, and transiently unwinds, a specific site in the origin, before difficult (e.g. a reduced negative superhelicity of the naked a helicase extends the unwound region. After recruitment of a template or a reduced temperature). The RNA polymerase does primase-polymerase complex, RNA-primed DNA synthesis not make primers as full activation still occurs when they are initiates on the leading strand. Next a processive polymerase terminated with 3′-dATP and so lack 3′-OH groups. Tran- captures the leading-strand primer and the primase-polymerase scription from one of the back-to-back promoters probably 1382 A. B. Hassan and P. R. Cook generates a RNA-DNA hybrid that stimulates origin for the viral T antigen (a dnaA analogue). Like the viruses unwinding (Baker and Kornberg, 1988). discussed above, an RNA polymerase II promoter with six Another example of helix-opening mediated by transcription Sp1-binding sites abuts and partly overlaps the origin core. is provided by phage λ (reviewed by Keppel et al., 1988). This overlap unfortunately compromises attempts to assess the Again, a promoter, PR, lies close to the origin and mutants with effects of transcription on replication by deleting the promoter. no transcription from PR generally activate new promoters that However, soluble extracts from monkey cells still replicate restore synthesis close to, but not necessarily across, the origin. exogenously-added origins even when transcription is elimi- And again replication in vitro under conditions that make nated by 250 µg/ml α-amanitin, an RNA polymerase inhibitor unwinding difficult depends upon transcription. As the cI (Li and Kelly, 1984). Moreover, replacing some or all of the protein represses both PR and lytic growth, the logic of this Sp1 sites with binding sites for different transcription factors circuitry ensures that replication quickly stops during lysoge- varies the transcription rate with relatively little effect on the nization. replication rate, unless multiple binding sites are present (Guo These examples show that the act of transcription can and DePamphlis, 1992; Hoang et al., 1992; Cheng et al., 1992). regulate initiation of replication in prokaryotes. In eukaryotes, A worry here is that the in vitro system that replicates oriC is the situation is less clear, largely because their origins are so also sensitive to an RNA polymerase inhibitor only under ill-defined (reviewed by Fangman and Brewer, 1992; DePam- certain conditions (see above), and the same may be true of the phlis, 1993) and no in vitro system is available that can initiate SV40 system. efficiently, other than a crude Xenopus extract (reviewed by Laskey et al., 1989). Fortunately, the origins of several viruses that grow in eukaryotic cells have been defined and systems EMBRYOGENESIS IN XENOPUS AND DROSOPHILA that replicate them in vitro established. Nevertheless, it is clear that, like their bacterial counterparts, eukaryotic origins are Embryogenesis in frogs and insects initially provided appar- always closely associated with transcription units. They are ently decisive evidence that initiation occurred in the absence also rich in binding sites for transcription factors and those of transcription. After fertilization, the Xenopus egg divides factors directly influence initiation (reviewed by Heintz, 1992; rapidly 12 times to give ~4000 cells; then, at the midblastula DePamphlis, 1993). What is unclear is whether binding alone transition, the rate of division slows (Newport and Kirschner, or binding plus transcription is necessary. The evidence will 1982a,b). No RNA synthesis, measured by incorporation of now be reviewed and its limitations highlighted. [3H]uridine (by whole cells) or [32P]UTP (after microinjec- tion), was initially detected prior to the midblastula transition. Furthermore, injecting α-amanitin to give an internal concen- TRANSCRIPTION FACTORS AND ORIGINS tration of ~50 µg/ml had no effect on the first 12 divisions but then prevented the appearance of transcripts generated by We begin with the incontrovertible evidence: origins bind tran- polymerase II and III. Similar results were also obtained with scription factors. For example, initiation at the adenoviral Drosophila eggs, which divide 10 times without detectable origin in vitro requires three viral proteins, including the DNA RNA synthesis or inhibition by α-amanitin (Edgar et al., 1986; polymerase, plus three host proteins; two of the latter turned Edgar and Schubiger, 1986). out to be transcription factors (CTF and OTF-1) and one binds These results imply that DNA can be duplicated without directly to the DNA polymerase (Jones et al., 1987; O’Neill et transcription but recent results provide some important caveats. al., 1988; Chen et al., 1990). Similarly, transcriptional trans- First, endogenous UTP pools, ~4 mM in Drosophila embryos activator proteins VP16 and GAL4 stimulate replication of (Edgar and Schubiger, 1986), inevitably dilute added radiola- bovine papilloma virus in vitro, again binding directly to a bel and make it difficult to detect transcription in the few nuclei replication factor (i.e. RPA; Li and Botchan, 1993; He et al., present earlier during embryogenesis. However, nascent frog 1993). The one chromosomal origin that has been analyzed in transcripts have now been detected at the 7th cleavage division molecular detail, yeast ARS1, also binds the transcription factor using higher concentrations of radiolabel (Kimelman et al., ABF1 and binding sites for other transcription factors (i.e. 1987). Moreover, some paternal Drosophila genes are RAP1 and GAL4) can substitute for the ABF1 site (Marahrens expressed at the third cleavage division (i.e. at the 8 cell stage) and Stillman, 1992). and so must be transcribed even earlier (Brown et al.,
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