ANINDYA DUTTA

Trans-plication factors?

DNA REPLICATION

_I. In addition to the known roles for transcription in the control of DNA synthesis, recent papers suggest that transcriptional activators may recruit a factor required for DNA replication.

Considerable insights i.nto the way eukaryotic DNA is replicated have come from studies of viral DNA replica- tion in intact animal cells or cell extracts. Mutagenesis of the viral genome identified the &acting DNA sequence elements that are requimd for replication in intact cells - the replication origin, 016, and neighbouring accessory se- quences. Similar studies have yielded information on how other cellular processes, such as transcription or passage through the cell division cycle, influence replication. The fractionation of animal cell extracts, on the other hand, has yielded the highly purified protein complexes that are essential for DNA replication in vitro. Subsequently, homologous factors have been identified in the budding yeast, Saccharomyces cerevisiae, the corresponding genes have been cloned, and genetic experiments in yeast have demonstrated that these factors are indeed essential for chromosomal DNA replication in vivo.

Among the eukaryotic replication factors that have been identified in this way is the complex named replication protein A (RFA) [1,2], which has single-stranded DNA- binding activity. The first step of DNA replication in vitro using ori from the papova virus SV40 (simian virus 40) occurs when the virally encoded origin-binding protein, T antigen, binds to ori. The next step, unwinding of the origin, requires RPA. Although T antigen untwists and melts the DNA double helix precisely at the replication origin, it cannot unwind DNA beyond ori itself without assistance from a single-stranded DNA-binding factor [ 31. Accumulation of sufficient WA at the origin is therefore

an essential early step in replication, although precisely how RPA is accumulated is uncertain. Does T antigen alone melt a region of duplex DNA that yields sulficient single-stranded DNA to recruit RPA (Fig. la)? Or is there a protein-protein interaction involved in the recruitment of RPA to the complex? A physical interaction has been demonstrated between T antigen and RPA [4], implying that cellular o&binding proteins that are functionally anal- ogous to T antigen - as yet unidentified in animal cells - could also interact with and recruit RPA (Fig. lb). Finally, an additional mechanism for recruiting RPA to the origin has recently come to light, which involves transcriptional activators and which may explain the role of accessory se- quence elements in DNA replication (Fig. lc). But before discussing this third mechanism I must provide a thumb- nail sketch of RPA. RPA is a complex made up of three polypeptides: 70 kD, 34 kD and 13 kD [5-71. The 70 kD subunit binds strongly to single-stranded DNA, a function believed to be essen- tial for the replication-promoting activity of RPA. However, the 70 kD subunit alone cannot support DNA replication in vitro: the 34 kD and 13 kD subunits are therefore likely also to have important functions. The genes encoding the three S. cerevisiae RPA subunits, RFAl’ (7OkD), RFA2 (34 kD) and WA3 (13 kD) have been cloned, and each has been shown by gene-disruption experiments to be essential for viability [ S,9], The terminal phenotype of yeast with disrupted RFA genes - a mother cell with a large bud attached - is consistent

(a) T anti en meets DNA, with t6e strands then binding RPA.

(b) T antigen itself binds RPA, separate B bringing RPA to the replication origin.

a’ ori

:c) Transcriptiotial activators ecruit RPA to the replication origin.

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Fig. 1. Alternative mod-& ?or the recruitment of RPA to a replication-initiation complex at the replication origin.

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~th a requirement for lthe RFA polypeptides in the tran- sition aron& s (DNA synthesis) phase of the cell cycle. The 34k~ subunit is plhosphorylated during the cell cy & h a stage-specific manner in both human and yeast cells, and it has been proposed that members of the Cdc2 cell-cycle regulatory kinase family might act at the Gl-to-S-p&se transition to phosphotylate and activate EPA [lo]. Phosphorylation of the 34 kD subunit of RPA has also been shown to be coupled with its binding to single-stranded DNA. at replication origins, although, in this case, phosphorylation is not due to Cdc2 kinase [Ill. RPA also interacts specifically with DNA polymerase ol-primase [ 12,131, and may therefore be involved in the assembly of a replication-initiation complex at the origin. In addition, EPA plays an important role in DNA repair and recombination; antibodies against EPA have been used to show that it is required for the excision-repair of dam- aged DNA [ 141, Its role in recombination was discovered when a proteolytic fragment of yeast RFAl was purified as a co-factor that stimulated a DNA-strand exchange re- action in vitro [8]. An unexpected facet of WA recently came to light with the discovery that it interacts with several transcriptional acti- vators [15,16]. It was already known that viral DNA repli- cation is stimulated by transcriptional activators, provided that binding sites for the activators are present close to the replication origin [17]. I and Botchan [ 151 were studying how replication of bovine papilloma virus (BPV) DNA was stimulated by a variety of transcription factors: E2 from BPV, and GAL4-VP16 and GAL4-p53 (both of which are recombinant transcription factors in which the DNA bind- ing domain of the yeast transcription factor GAL4 is fused to the acidic transcriptional activation domain of either the herpes virus protein VP16 or the the human tumour sup- pressor protein ~53). Li and Botchan [15] observed that the activation domain of VP16 was able to deplete from cell extracts a factor essential for BPV DNA replication, and found that the depleted factor was EPA. He et al. [l6] analyzed cellular proteins that bound to a column of immobilized VII6 acidic domain, in or- der to dissect the mechanism by which ~~16 activates transcription and replication. Sequencing one of the ma- jor polypeptides bound to the column identified it as the 70kD subunit of RI%. A point mutation in the ~~16 ac- tivator domain that destroys its transcription-stimulating activity also abrogated both its ability to stimulate polyoma virus DNA replication and its capacity to bind to EPA. Both groups extended their results by showing that other acidic activator domains, such as those from the tumour sup- pressor p53 or the yeast transcription factor GAL4, also interact with the 70kD subunit of EPA These ftndings led to the hypothesis that accessory replication sequences, located close to ori sequences, bring transcriptional acti- vators close to the origin; the acidic activation domains then promote DNA replication by recruiting RPA through protein-protein interactions (Fig. lc). Transcription factors ‘have been implicated in DNA repli- cation in many previous experiments. For example, transcriptionally active regions of the chromatin are

replicated earlier in S phase than transcriptionally inac- tive regions [ 181. In addition, two cellular factors iden- tilied as essential for adenovirus DNA replication, NFI and NFIII, can also function as transcription factors, in which case they are known as the CCAAT box binding factor and Ott-1, respectively [19,20]. Viral minichromo- somes are replicated more efficiently when they contain transcriptional enhancers that bind to transcription fac- tors present in the host cell than when they do not [17]. And in u&o, in the SV40 viral replication system, transcriptional activators stimulate DNA replication if the DNA-binding sites for the activators are present near the replication orlgln [21,22]. Finally, ln S. cerevisiae, an ac- cessory sequence next to the core ori within the larger c&-acting autonomous replication sequence (ARS) that is required for DNA replication has a stimulatory effect on DNA replication only when bound to the yeast transcrip- tion factor ABFl [ 231; the binding site for a heterologous transcription factor can also serve as an accessory replica- tion element provided that the heterologous factor is also present in the yeast.

Acti

’ Active RPA 0 1993 Current Biology

Fig. 2.‘Activation of the DNA-binding function of RPA after recruitment to an origin of WA replication. (a) and (b) denote alternative pathways (see text).

There are several theories to explain how transcription factors activate DNA replication. Experiments with the SV40-based in vitro replication system suggest that a major effect of these transcription factors is to alter the chromatin near to the replication origin such that replica- tion factors have easier access to the origin [ 221. A second mechanism can be envisaged by analogy with prokaryotic replication systems, in which transcripts from promoters adjoining replication origins are extended into the origin area and can stimulate replication either by providing primers for DNA polymerase or by making it easier for replication factors to melt the origin [24]. The new ob- servations [15,16] that transcription factors bind directly to EPA provide a third possible mechanism: the activators could recruit an essential replication factor to the ori- gin and thus promote replication, This is analogous to the mechanism by which transcriptional activators are believed to promote transcription, the key differences between the two processes lying in the type of factor recruited - a replication factor instead of transcription

_. DlSPATdti 711

Acknowlwedgements: I thank Ellen Winchester, Clark Chen, I.orene Leiter, Jon Aster and Kylie Keshav for illuminating discussions and advice.

factors such as ‘I’ATA-binding protein, TFIIB or TAF,,, - and in the DNA element that lies near the activator-binding site - a replication origin rather than a transcriptional promoter.

My colleagues and I have been interested in the mech- anism by +&ich the tumour suppressor protein and transcriptional activator, ~53, inhibits the onset of S phase. We have recently shown that ~53, in part through its transcriptional activation domain, binds to the amino- term inal part of the 70 kD subunit of RPA and inhibits the ability of RPA to bind to single-stranded DNA [25]. This raises the following question: do all transcriptional activators also inhibit the DNA-binding activity of RPA? If they do, then in order for these activators to serve as accessory factors for ‘DNA replication there must be a mechanism to relieve RPA from inhibition. For ex- ample, in the case of p53, specific phosphorylation of ~53, which occurs at the Gl-S-phase boundary, could be involved in dissolving the p53-RPA complex (Fig. 2a). Alternatively, an origin-binding protein (such as T antigen) could interact with RPA and recruit it away from the adjoining FPA-transcription factor complex and into the replication-initiation complex in an inactive form (Fig. Zb).

But the inhibition of RPA DNA-binding activity m ight be a unique property of p53 -- the other transcriptional acti- vators that affect replication may instead recruit function- ally active RF’A to the replication origin. In that case, the RPA-p53 interaction may be important in allowing p53 to inhibit the onset of DNA replication at the Gl-S-phase transition [26,27], or to impose a delay to allow DNA repair after radiation-ind’uced DNA damage [ 281. Trans- form ing mutants of ~53, however, which neither inhibit the onset of S phase nor delay DNA replication following DNA damage, nevertheless interact with RPA and inhibit its origin-binding activity in vitro. If the WA-p53 interac- tion is important for ~53 to inhibit DNA replication, one must postulate that the transform ing mutants of p53 fail to bind to RPA in vivo, due perhaps to aberrant subcellular compartmentalization or to interaction with other cellu- lar proteins. It is unclear what effect, if any, the phospho- rylation of RPA has on the interaction between RF’A and transcription factors.

Finally, the ability of RPA to interact with transcriptional activators m ight indicate that RPA has a role in the reg- ulation of transcription. A single-stranded DNA-binding protein m ight indeed hzwe a function in the formation of an ‘open complex’ at eukaryotic transcription promoters. Transcriptional activators that are known to interact with basal transcription factors by way of acidic activation do- mains have now been shown also to interact with a basal replication factor using lthe same domains, The interplay between transcription and replication that has long been apparent can now be explained. Clearly, many questions are raised by these new findings, but the answers to sev- eral of them should&rLve in the next few months, illu- m inating the cross-talk$etween transcription, replication, and perhaps tumour“&pression.

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muy q: Transxiption factor QTF-1 is functionalLy iden- tical to the DNA replication factor NF-III. Science 1988, 241:1210-1213.

21. C~NG L, KELLv TJ: Transcriptionti activator nuclear factor I stimulates the replication of SV40 minichromosomes in uiuo and in vitr0 Cell 1989, 59541-551.

22. CHE~G IZ, WORKMAN J& KINGSTON RE, KELLY TJ: Regula- tion of DNA replication in vitro by the transcriptional activation domain of GAL4-VPl6. Pm Nat1 Acad Sci USA 1992, 89589-593.

23. MARAHRENS Y, %LLMAN B: A yeast chromosome origin of repli- cation defined by multiple functional elements. Science 1992, 255:817-823.

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Anindya Dutta, Department of Pathology, B&ham and Women’s Hospital and Harcrard Medical School, 20 Shattuck Street, Boston, Massachusetts 02115, USA.

IN THE OCTOBER 1993 ISSUE OF CURRENT OPINION IN GENETICS AND DEVELOPMENT

Elizabeth H. Blackburn and Lucy Shapiro edited the following reviews on the Regulation of Chromosome Dynamics in Microorganisms:

Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP by Arthur Landy

Protein-DNA iassemblies controlling lytic development of bacteriophage Mu by Tania A. Baker The retron: a bacterial retroelement required for the

synthesis of msDNA by Sumiko Inouye and Masayori Inouye SOS mutagenesis Sumati Murli and and Graham C. Walker

Restructuring of DNA sequences in the germline genome of Oxytricha by David M. Prescott Developmentally regulated processing and replication of

the Tetra@mena rDNA minichromosome by Geoffrey M. Kapler Meiotic recombination in yeast by Catherine L. Atcheson and Rochelle Easton Esposito

Lineage-dependent mating-type transposition in fission and budding yeast by Amar J.S. Klar The structure and function of yeast ARS elements by Carol S. Newlon and James P. Theis

Telomeres and telomerases by Dorothy E. Shippen Tmnscription and DNA supercoiling by James C. Wang and A. Simon Lynch

Conserved features of yeast and mammalian mitochondrial DNA replication by Mark E. Schmitt and David A. Clayton

Bacterial chromosome origins of replication by Gregory T. Marczynski and Lucy Shapiro Escberichia coli cell division by Joe Lutkenhaus

Chromosome partition in Escherichia coZZ by Sota Hiraga