checkpoint control, and transcription

A Coordinated Interplay: Proteins with Multiple Functions in DNA Replication, DNA Repair, Cell Cycle/ Checkpoint Control, and Transcription MANUEL 100~

STUCKI,

STAGLJAR,

ZOPHONIAS ULRICH

0. JONSSON,

AND

H~~BSCHER~

Department of Veterinary Biochemistry University of Ziirich-lrchel CH-8057 Ziirich, Switzerland ,.............,,.,....... I. DNA Polymerases . A. DNA Polymerase cx ... . . B. DNAPolymerasesSande .................................... II. DNA Polymerase Accessory Proteins .............................. A. ReplicationFactorC ......................................... B. Proliferating Cell Nuclear Antigen ............................. III. Transcription Factors and Their Role in Activation of DNA Replication A. Introductory Remarks ........................................ B. Lessons from Viral Systems ................................... .......................................... C. LessonsfromYeast D. How Might Transcription Factors Help in Activating DNA Replication? ...................................................... IV Perspectives and Conclusions .................................... References ....................................................

In eukaryotic scription require

cells, DNA

transactions such as replication,

263 263 271 279 279 281 286 286 286 287 290 291 292

repair, and tran-

a large set of proteins. In all of these events, complexes of more

than 30 polypetides

appear to function in highly organized

and structurally well-

defmed machines. We have learned in the past few years that the three essential macromolecular

events, replication, repair, and transcription, have common fimc-

tional entities and are coordinated by complex regulatory mechanisms. This can he documented

for replication and repair, for replication and checkpoint control, and

for replication

and cell cycle control, as well as for replication

’ To whom correspondence Progress in Nncle~c Acid Research and Molecul;ur Riology, Vol. 65

and transcription.

should be addressed.

261

Copyright 0 2001 by Academic Press. All rights of reproduction in any form resewed. OOiY-6603Lll $35.00

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In this review we cover the three different protein classes: DNA polymerases, DNA polymerase

accessory proteins, and selected transcription factors. The “common

enzyme-different

pathway strategy” is fascinating from several points of view: fmt,

it might guarantee that these events are coordinated; an evolutionary

second, it can he viewed from

angle; and third, this strategy might provide

mechanisms for essential physiological tasks.

6 zoooAcademic

cells with backup

press.

Maintenance of genetic stability is a key issue for any form of life. Consequently highly sophisticated mechanisms for maintenance of the genome were well established before the three kingdoms of life (Archaea, Eubacteria, and Eukaryotes) separated. DNA replication is the event leading to the duplication of DNA in advance of mitosis (or meiosis) and cell division. It occurs in vivo in an ordered and highly organized way, in which all enzymes and proteins involved have their exact roles in a replication complex called the replisome, which is located in so-called nuclear replication factories. Models have been proposed on how the enzymatic machinery might be spatially arranged at replication forks (I). The models were based on the idea that DNA polymerases (~01s) dimerize and that the DNA forms loops on the lagging strand such that the “directionality” for the pol is the same. If one postulates that the replisome is fixed to structures in the nuclear replication factories, it would thread the DNA through itself. The assembly and the events in a replisome might occur as follows (2): 1. An initiator protein complex, the origin recognition complex (ORC), is bound to an origin of replication. The ORC has to be activated by other proteins, such as minichromosomal maintenance (MCM) and cell division cycle (Cdc6 and Cd7/Dbf4) proteins, by mechanisms such as phosphorylation or possibly other posttranslational modifications. This leads to the formation of an initiation complex that is able to alter DNA structures in its vicinity, presumably by activating the intrinsic helicase activity of an MCM hexamer or by attracting other DNA helicases to the origins. The single-stranded DNA thus produced must be protected and stabilized by the single-stranded DNA binding protein, called replication protein A (RP-A); RP-A can help to unwind the DNA by its unwinding activity and, possibly, through its interactions with DNA helicases and pol (Yprimase (pal (Y),which acts as the initiating pol. 2. After very limited DNA synthesis a DNA polymerase switch from pol (Y to the processive ~016 holoenzyme occurs, most likely mediated by the pol auxiliary protein replication factor C (RF-C). The pol (Ycomplex subsequently acts at the discontinuously synthesized lagging strand, where

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it initiates Okazaki fragments. While the pol S holoenzyme [pol 6, proliferating cell nuclear antigen (PCNA), and RF-C] is engaged in processive leading-strand DNA synthesis, the situation at the lagging strand is more complex. A second pol (pal S or E) holoenzyme is formed for completion of each Okazaki fragment initiated by pol (Y.DNA syntheses on the leading and on the lagging strand are perhaps coordinated by dimerization of the two processive pol holoenzymes, possibly on physical interaction of two pols or via a clamp factor. 3. The initiator RNA at the lagging strand is removed by RNase H and by a 5’ -+ 3’ exonuclease such as flap endonuclease (Fen 1) or by the Dna2 endonuclease. After complete synthesis the Okazaki fragments are sealed by DNA ligase I (Lig I). Topological constraints are released by DNA topoisomerase I and the replicated DNA can finally be separated by DNA topoisomerase II. Duplication of the genetic information is not the only DNA transaction. Integrity of the genome in nondividing cells is maintained by various DNA repair mechanisms (3), including nucleotide excision repair (NER) (4), base excision repair (BER) (5), mismatch repair (MMR) (6), and double-strand break repair (DSBR) (7). Th ese mechanisms, together with control processes such as checkpoint control (8), guarantee that either newly replicated DNA goes without mutation into mitosis or that nondividing cells maintain the DNA at a level of mutations that guarantees proper function. Most of the various DNA repair processes and DNA replication might not function in an “authistic” way, but rather are connected to each other. Controlled transcription is the event that fullfils the requirements for a timely and coordiated expression of the genetic information to yield proteins with specific functions. Transcriptional events have also been found to be of importance for initiation of DNA replication. In the following three sections we focus on multiple functions of the three pols, (Y,6, and E; the two pol auxiliary proteins, RF-C and PCNA; and on the four transcription factors, Ga14, ~53, BRCAl, and ABFl, which can function in DNA replication via their activation domains (see Table I).

I. DNA Polymerases A.

DNA

Polymer-use 01

1. GENERAL DESCRIPTIONOF THE ENZYME In 1957 the first eukaryotic polymerase, pol (Y,was discovered (9). Pol (Y still holds a special position in the growing family of eukaryotic pols, because it is the only enzyme that can start DNA synthesis de nowo. It first synthesizes

TABLE

I

break

see text.

Double-strand

“PO1a/plimase complex.

“DSBR,

aFor references,

Gal4

repair;NER, nucleotide

+ +

+ + + +

P53 BRCAI ABFl

excision repair; BER, base excision

+ + -

-

repair;MMR,

mismatch

repair.

+ + + +

Apoptosis Recombination _

140.kDa subunit; binds to telomerase _

_

-

3 7-kDa subunit

-

NER, BER, MMR, DSBR NER, BER, MMR, DSBR

PCNA RF-C

_

_

C terminus of large subunit

NER, BER, DSBR

PO1E

Other functions Telomer length (2) -

Primase ?

DSBR NER, BER, MMR, DSBR

Initiating pol Leading-s&and pal; lagging-strand pol? Leading strand pol ?; lagging-strand pol + +

Pol a” PO16

Transcription _ _

DNA repa?

DNA replication

Protein

Checkpoint control

Functior8’

PROTEINSWITHMULTIPLEFUNCTIONS”

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265

an RNA primer of about 10 nucleotides followed by extension of the primer to produce a short piece of DNA. Therefore, the major task of pol (Yis thought to be the initiation of DNA synthesis during replication. The human enzyme is composed of four individual subunits with molecular masses of 180, 70, 58, and 48 kDa, and a similar subunit composition was found for all eukaryotes examined (IO). The DNA polymerizing activity is located on the largest (~180) subunit whereas the p48 subunit contains the catalytic center of the primase function. Although isolated ~48, devoid of any detectable p58 subunit, is sufficient for RNA primer synthesis, primase activity associated with free ~48 is highly unstable, indicating that its association with the other polypeptides of the pol LXcomplex is crucial for stabilizing the enzyme activity (11). The 180-kDa subunit exists as a tight complex with the p70 subunit and it was found that this interaction is essential for both the synthesis of the ~180 protein and its translocation into the nucleus (12). No catalytic activity was found in p70 subunit per se, but it is thought to play an important role in modulating the activity of the whole complex as well as in controlling its ability to associate with the chromatin at the origins of replication. This guarantees a tightly controlled and coordinated initiation of DNA synthesis on both the leading and the lagging strand. These effects are most probably mediated through cell cycle-specific phosphorylation of pol (Y and/or interaction with other protein partners that are involved in DNA metabolism (summarized in Table II). 2. ROLE OF POL (YIN VIRAL DNA REPLICATION Biochemical studies based on plasmids containing the simian virus 40 (SV40) origin of replication have uncovered the fundamental mechanisms leading to the recognition of the origin sequence, the local unwinding of the DNA, and the loading of the pol (Ycomplex onto the DNA. Only two proteins, the virus-encoded SV40 large T antigen (TAg) (13) and RP-A, are required. They interact with each other and with pol (Y,tether the pol to the DNA, and cooperate to initiate DNA synthesis at the SV40 origin. (13, 14). Data obtained using the human papillomavirus (HPV) replication system suggest a similar mechanism of initiation. The HPV El helicase together with the E2 protein bind cooperatively to the viral origin of replication, forming an ElE2-ori complex similar to the SV40 TAg-ori complex. The El protein interacts with RP-A and with the ~180 and p70 subunits of pol cx and might bring pol cx to the DNA in a way similar to that of TAg (15-17). As soon as pol (Yis associated with the initiation complex, it synthesizes a short RNA-iDNA (i stands for initiator) fragment, approximately 10 nucleotides of RNA followed by 30 nucleotides of DNA, which then serves as a primer for extension by another pol(18 -21). This process (called polymerase

II

Viral and cellular DNA replication; primosome assembly ? Lagging-strand synthesis? Lagging-strand synthesis? ? Okazaki fragment processing Cell cycle regulation Cell cycle regulation Cell cycle regulation Tumor suppressor, DNA replication; proofreading? Tumor suppressor Tumor suppressor DNA damage survey?

Human Calf Mouse S. s. S. s. s. cerevisiae; xenopus Human Human s. cer-evisiae Human Human Human; mouse Human

pi; fi

pi; fi

pi; @

pi pi pi

pi; fi

fi fi fi

pi pi pi

pi

RP-A

AAF

Cdc68p/SptlGp PoblpKtf4p Pob3p Dna2p

Cdc45piXCdc45p

Cyclin A-Cdk2p Cyclin E-Cdk2p Cdc28p-Clb

P53 Rb Doe-1

PARP

aFor references, see text. bAbbreviations:Ag, Antigen; AAF, alpha accessoryfactor; Rb, retinoblastomaprotein; PARP, poly(ADP)ribose polymerax “pi, Physical interaction;fi, functional interaction; gi, genetic interaction.

Primosome assembly

Viral DNA replication; primosome assembly Viral DNA replication; primosome assembly

Simian virus 40 Papillomatis

pi; fi pi; fi

SV40 large T Ag El

cerevisiae cerevisiae cerevisiae cerevisiae

Presumed functional task with pol u

Species

U/fiIMASEa

Type of interactionC

WJTH POL

TABLE hTERACTINC

Protei&

PROTEINS

PROTEINSWITHFUNCTIONSINDNAPROCESSES

267

switch) is not yet fully understood. Although SV40 or&dependent replication in vitro with highly purified enzymes showed that pol cx is only moderately processive (22), it is unlikely that the limited size of the RNA-iDNA primer synthesized by pol (Yin vivo is the result of a spontaneous dissociation of the enzyme from the nascent DNA due to its lack of processivity. Indirect evidence suggests that the clamp loader RF-C plays an important role for the polymerase switch (21, 23) and additional results have confirmed an active role of RF-C in this process (see Section II). On the leading strand, pol (Yhas to initiate DNA synthesis only once per round of viral replication, whereas on the lagging strand, it has to reinitiate DNA syntheis for every Okazaki fragment. This poses a problem, because pol (Ydoes not have a proofreading activity and thus is unable to remove any errors that it inserted into the iDNA on the lagging strand. Although these mismatches might cause only minor implications for viral replication, they must be repaired on the over 20 million Okazaki fragments initiated during one round of cellular DNA replication (in human cells). One interesting and yet unsolved question is why pol o. contains a DNA pol fuction at all, because the other replicative pols, S and E, are able to elongate RNA primers in vitro (24). However, mutation analysis in yeast revealed that an operative pol fuction is essential (25). Ob viously, the cell is somehow able to deal with this problem. 3. POL 01MIGHT BE PARTOF A MULTIPROTEINCOMPLEX FORCOORDINATEDANDERROR-FREE LAGGING-STRANDREPLICATION

It has been proposed that the synthesis of an RNA-iDNA primer to start an Okazaki fragment and maturation of the previous Okazaki fragment are coordinately carried out during lagging-strand synthesis by a multiprotein complex, and that this complex might contain all the required enzymatic functions to remove RNA-iDNA primers that contain mismatches (14, 26, 27). Studies now support this hypothesis: mutations in the essential DNA2 nuclease-helicase gene from the yeast Saccharomyces cerevisiae were found to interact genetically with POLl and CTF4/POBl, which encode the large subunit of yeast pol cx and a protein involved in DNA metabolism in vivo (28). DNA2 has previously been shown to interact genetically and physically with RAD27, the yeast homolog of the human structure specific nuclease Fen 1 (29) that is involved in Okazaki fragment maturation. It has been proposed that Fen 1 might be able to remove the mismatch containing iDNAs endonucleolytically (2 7). H owever, Fen 1 does not act here as a single player because the gene is not essential in yeast. There is growing evidence that the task of quick and error-free synthesis as well as maturation of the Okazaki

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fragments are carried out by a complex multiprotein machinery containing at least pol (Y, pol 6, PCNA, RF-C, RP-A, Dna2, Fen 1, and Lig I (K.-S. Seo, personal communication). This suggests that the iDNA could be replaced by rereplication of the resulting gap by one of the proofreading pols, 6 or E. Such multiprotein complexes have been isolated from S. cerevisiae (30) and calf thymus (23). 4. ROLEOFPOL~YINTHECELLCYCLECONTROL OFDNAREPLICATION Most of the mechanisms of replication fork progression have been studied with viral in vitro replication systems such as SV40. Therefore, much less is known about cellular DNA replication. Nevertheless, in recent years, genetic studies using the yeasts S. cerevisiae and Schizosaccharomyces pombe, as well as in vitro and in vivo protein-protein interaction studies, have provided exciting insights into the complex network of biochemical signaling pathways that control chromatin replication and couple it tightly to the S phase during the cell cycle (31). The unique ability of pol (Y to initiate DNA synthesis makes it a perfect downstream target for cell cycle control pathways. It has been observed that polo is phosphorylated in a cell cycle-specific manner in yeast (32) and human (33) cells. Most of the phosphorylation occurs late in the cell cycle, suggesting that it does not play a role for initiation control. However, phosphorylation of both the ~180 subunit and the p70 subunit could also be achieved in vitro, using cyclin-dependent kinases (cdks). This phosphorylation did not influence the pol activity on primed templates, but it slightly stimulated primase activity, and specific initiation of replication on SV40 ori-containing plasmids was affected. Cyclin A/Cdk2 inhibited the ability of pol (Y to initiate SV40 DNA replication, whereas phosphorylation by cyclin E/Cdk2 stimulated its initiation activity (34). Tiyptic phosphopeptide mapping of the in vitro phosphorylated p70 subunit and p70 from human cells that were synchronized and labeled in G,S and in G, showed similar patterns: a cyclin E/ cdk2-like pattern in G,/S and a cyclin A/cdk2-like pattern in G, (35). This suggests that replication activity of pol (Yis regulated on phosphorylation by cdks in vivo. Another important regulatory pathway that is mediated through cdks limits DNA replication to once per cell cycle because these kinases can also prevent rereplication. Assembly of prereplicative complexes at origins can occur only during G,, because assembly is blocked by cdkl together with B cyclins (Clbs). Desdouets et ~2. describe the recruitment of pol (Yto chromatin as a second separate process that can occur only during G,, because recruitment is also blocked by cdkl/Clb, indicating that this might be an additional layer of control (36).

PROTEINS

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Several proteins have been found to interact either physically or genetically with pol OL(see Table II). Among these, some are thought to modulate pol (Y activity in a cell cycle-specific manner or support loading of the complex onto chromatin. Almost 10 years ago a protein isolated and purified from mouse cell extracts was called alpha accessory factor (AAF), because it was able to stimulate pol (Y activity severalfold on synthetic templates (37, 38). However, its physiological function remains unclear. CDC68SPT16, a gene from S. cerevisiae, is required for passage through the cell cycle control point START, and Cdc68p was shown to bind to pol (Yby affinity chromatography (39). The same method has led to the identification of two other proteins, PoblpKtf4p (40) and Pob3p, a protein with significant amino acid similarity to a HMGl-like protein from vertebrates. Cdc68p and Pob3p seem to compete with Ctf4p for binding to pol CLCDC68 also interacts genetically with POLl and CTF4, indicating that the proteins are involved in the same biochemical pathway (39). Interestingly, a CDC68 mutation was also synthetic lethal with a DNA2 mutation, suggesting that the proteins may rather be part of a complex that is responsible for coordinating lagging-strand DNA syn thesis (see above), rather than supporting recruitment of pol o_to the origins. Moreover, another protein, called Cdc45p (XCdc45), has been identified in budding yeast and Xenopus egg extracts. It is essential for the initiation of replication and has been shown to interact physically with pol CLThere is growing evidence that this protein plays a pivotal role in the loading of pol (Y onto chromatin under the control of S phase cdks (41). Finally, three human tumor suppressor proteins have been shown to interact physically with pol ct as well: p53 (42), DOC-1 (43), and the human retinoblastoma protein Rb (44). The interaction between pol OLand p53 is of particular interest, because p53 is believed to have an intrinsic 3’ + 5’ exonuclease activity and colocalizes with DNA synthesis in uiuo. It has been suggested that p53 might increase pol (Yfidelity during replication (45, 46). 5. POLCIASAPOTENTIALPART

OF

CELLCYCLE

CHECKPOINTPATHWAYS Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. In addition, checkpoints respond to DNA damage by either arresting the cell cycle or slowing down S phase to provide time for repair and by inducing transcription of genes that facilitate repair (8, 47). In S. cerevisiae, two essential genes from the central conduit for checkpoint signal transduction were identified: MECI (related to the ATM gene that is defective in the human disease ataxia telangiectasia) and RAD53; both encode protein kinases.

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The first evidence that pol OLcould be involved in one or several checkpoint pathways came from genetic studies with the yeast S. pombe. The gene cd.cl +, the homolog of RAD53, has been identified as a multicopy suppressor of a temperature-sensitive mutant in the pol (Y gene (48). Another group showed that germinating S. pombe spores, disrupted for the POLl gene, could enter mitosis despite defects in DNA synthesis (49). Possibly pol 01 is required for a checkpoint signal that is activated as cells traverse START, and is essential to prevent mitosis until S phase has been completed. Moreover, genetic studies with S. cerevisiae revealed that the primase function of pol (Y might be a final target for the S phase checkpoint. A temperature-sensitive mutation in the primase gene was defective in DNA synthesis at the permissive temperature in the absence of DNA-damaging agents, whereas the same mutant, in the presence of the DNA-damaging agents proceeded faster through S phase than did wild type (50). An explanation for this apparent paradox could be that this mutant somehow fails to respond properly to a regulatory mechanism that is required to inhibit G, + S transition and S phase progression in the presence of DNA damage. The primase may be inhibited on DNA damage in order to prevent reinitiation of DNA synthesis downstream to the lesion, which then could lead to subsequent slowing down of S phase progression. Interestingly, mutations in the MECl gene were shown to result in a similar phenotype (51), indicating that yeast pol cx could indeed be part of the Meclp checkpoint pathway. Finally, the yeast protein Cdc45p, which is believed to have a key regulatory function for association of pols onto the origins (see above), is downregulated on induction of the S phase checkpoint, revealing evidence that the loading of pol OLonto chromatin is an additional target for checkpoint regulation (52). 6. Is POL a INVOLVEDIN DNA REPAIR? Bulky DNA lesions such as cyclobutane pyrimidine dimers or 6-4 photoproducts are efficiently repaired by nuclear extracts from Xenopus oocytes. This repair process seems to be dependent on pol CY,because neutralization of the enzyme with a specific antibody resulted in reduced repair effeciecy that could be restored by the addition of purified pol (Y(53, 54). However, a direct role of pol (Y in one of the excision repair pathways has not yet been reported. A novel model for double-strand break (DSB) repair has been proposed (55). Holmes and Haber analyzed mitotic DSB-induced gene conversion at the MAT locus in mutant S. cerevisiae strains that were temperature sensitive for essential replication factors. Surprisingly, a pol (Y mutant turned out to be greatly defective for completion of gene conversion at the MAT locus, indicating that lagging-strand synthesis is required for this process.

PROTEINS WITH FUNCTIONSIN DNA PROCESSES

B. DNA

Polymerases

271

6 and E

1. GENERAL DESCRIPTION OF THE ENZYMES Pol6 and pol E share two common features: they contain intrinsic 3’ -+ 5’ exonuclease functions that contribute a useful proofreading activity and they are responsive to the accessory proteins PCNA and RF-C (see also Section II). Pol S is thought to be the major replicative and repair pol(56, 57). Its activities have been characterized extensively by in vitro studies using the SV40 replication system and broad genetic and biochemical studies were carried out with the yeasts S. cerevisiae and S. pombe. The subunit composition of pol 6 is complex and remains ambiguous. Most likely, the S. cerevi.siae enzyme is composed of three individual subunits with apparent molecular masses of 125, 58, and 55 kDa, encoded by the genes POL3 (or CDC2), PO1531 (or HYSB), and POL32, respectively, which may form a dimer or a heterotrimer (58). However, an active heterodimeric form composed of Pol3p and Po13lp that showed a biochemical behavior somewhat different than that of the trimer could be isolated as well (59). Furthermore the POL32 gene is not essential for viability in budding yeast. In contrast, ~016 from S. pombe seems to be composed of at least four, and perhaps even five, subunits that migrate on sodium dodecyl sulfate (SDS) gels; the subunits have molecular masses of 125 (PO/~+), 55 (C&l+), 54 (C&27+), and 42 and 22 kDa (Cdml’) (60). The mammalian enzyme has been isolated and characterized from various sources. Highly purified preparations were described as a two-subunit enzyme, with a 125-kDa subunit (~125) and a subunit from calf thymus (24, 61) and mouse cell extracts (62) that was 48-50 kDa (~50). The genes of these two subunits have been isolated and cloned from mouse, calf, and human (56) cells; although the gene of the largest ~016 subunit shows high homology throughout the species, the genes for the small subunits are much less conserved. The ~125 subunit carries the catalytic centers of both pol and exonuclease functions whereas the functional tasks of the small subunit(s) are not understood. Mouse ~018 can be purified in two forms: the single catalytic subunit that is not responsive to PCNA and the two-subunit enzyme that is stimulated by PCNA (62). Similar observations were made with highly purified pol 6 fractions from calf thymus and HeLa cells (M. Stucki and U. Hiibscher, unpublished results) and for recombinant ~125 from either Es&et-i&a coli (63) or from recombinant baculovirus-infected insect cells (64). A study using PCNA affinity chromatography as an alternative approach to search for additional subunits provided more evidence that mammalian ~018 is indeed composed of more than two subunits. A new polypeptide from a mouse cell line extract had an apparent molecular mass of 66 kDa and

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MANUELSTUCKIETAL.

coeluted from a PCNA affinity column with ~125 and ~50. Sequence characterization and a sequence data base search led to the identification of an open reading frame, and the carboxy-terminal part of the corresponding clone showed significant homology to the carboxy terminus of C&27+ from S. pombe, although overall homology between these two sequences is low (65). Assessing all of these data, it seems that mammalian pol 6 can be isolated in at least three forms: as a monomer (p125), as a dimer (~125 together with p50), and as a multisubunit complex that contains at least three, perhaps more, polypeptides. However, the question of a physiological significance of these subforms has not yet been addressed and therefore an exact characterization of the components of the mammalian ~016 complex and their biochemical roles is still elusive. Pol E is the second PCNA-responsive pol. Four to five subunits have been identified for S. cweuisiae pol E. The genes POL2, encoding the large 256kDa catalytic subunit, and DPB2, encoding one of the small subunits (80 kDa), are essential (66, 67), wh ereas the genes DPB3, encoding one or two subunits of 34 and 3 1 kDa, respectively, and DPB4, which encodes the fourth 29-kDa subunit, are not essential (68). The S. pombe gene Cdc20+ encodes the catalytic pol E subunit of fission yeast (70). In human cells the enzyme consists of two polypeptides of 2 15 and 55 kDa (71), whereas it was purified from calf thymus as a complex of 140, 125, 48, and 40 kDa (72). The 140kDa polypeptide is most likely a degradation product of a large precursor, because the enzyme has a high tendency to degrade during purification procedure due to a protease-sensitive site within the large catalytic subunit. However, the degraded 140-kDa form contains all the catalytic fuctions required for DNA synthesis and proofreading activity (73). Pol E genes from some mammalian cells have been cloned and characterized (74, 75). 2. THEPOL~ANDPOLEHOLOENZYMES The complex of ~016 or pol E with PCNA and RF-C is referred to as the holoenzyme form of the corresponding enzyme (76- 78). Assembly of the holoenzyme at a template-primer junction and pol switching are most probably orchestrated by RF-C (see Section II), which might leave the complex after assembly of a processive pol-PCNA clamp (79). The exact architecture of these processive pol machines is complex and has not yet been resolved to molecular details. The pol activities of pol6 and pol E appear to depend strongly on the geometry of the template (80) and on the assay conditions. On a linear primed homopolymer poly(dA)-oligo(dT)] pol E is fully processive in the absence of PCNA, whereas on the same template pol S is processive only in the presence of PCNA and under low-pH conditions. In this case, RF-C is not required because PCNA is able to slide

PROTEINS

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on the linear DNA spontaneously. On primed circular templates, however, both pols form stable processive holoenzyme complexes only in the presence of RF-C, PCNA, ATP, and a single-strand binding protein such as RP-A or E. cc& SSB (81,82). Th e same requirement for accessory factors can be achieved on linear DNA in the presence of ATP and under physiological pH conditions (83). One important difference between ~016 and pol E holoenzymes was reported for gap-filling synthesis (84). On double-stranded DNA circles containing a defined gap, pol 6 and pol E in the presence of PCNA and RF-C could fill the gap, and pol S, but not pol E, was able to synthesize up to 150 nucleotides into the double-stranded region (84, 85). This observation, called “limited strand displacement activity,” could have strong implications for the pol usage either for Okazaki fragement processing or for gap-filling synthesis during repair such as NER (86, 87) or BER (88). The mechanism of how PCNA stabilizes the pol-template complex, thus transforming the distributive enzyme into a processive form, is not yet clear. Several studies report a direct physical interaction between the N terminus of mammalian ~016 ~125 to the so-called domain-connecting loop of PCNA (89- 92). However, this interaction does not stimulate, or only slightly stimulates, processivity of the ~125 subunit alone. The ~50 subunit seems to be required for full stimulation (64), although this protein most probably does not interact directly with PCNA (92). S accharomyces cerevisiae pol &PCNA interaction has been confined to the Po132p subunit using both the yeast twohybrid system and a PCNA overlay method (58), but deletion of the gene for this subunit is not a lethal mutation (see above). Moreover, the question of the relative positions of components of the mammalian pol S/PCNA complex has been addressed: it seems that PCNA is located “behind” ~016 in regard to the DNA synthesis (3’ -+ 5’) direction and that only the large catalytic subunit contacts the DNA (93). Taken together, these data suggest that at least one (perhaps more) of the pol 6 subunits directly contacts PCNA and that this interaction stabilizes the enzyme on the DNA and might also induce a conformational change of ~125 and p50 relative to each other, which could be crucial for the transformation of the catalytic subunit into a processive form. The biochemical impact of PCNA on ~016 is different from that on pol E. First of all, pol E is processive on primed linear homopolymer DNA in the absence of PCNA. However, under conditions of high ionic strength, pol E becomes highly dependent on PCNA (94). Exact kinetic analysis revealed that PCNA is able to increase both the affinity for the primer and the rate of nucleotide incorporation by pol E, thus stimulating its activity (95). By using four characterized mutants of PCNA (91) containing three to four alanine

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residue substitutions, either on the C-terminal side (which is located on the “front side” in regard to the DNA synthesis direction) or on the back side of the trimer, the kinetics of primer binding and nucleotide incorporation by pol E were tested on linear DNA (96). In contrast with what had been found in interaction studies between pol S and PCNA, these data suggested that stimulation of pol E primer binding involves interactions with both the C-terminal side (frontside) and the backside of PCNA, whereas for stimulation of pol E DNA synthesis, exclusively the C-terminal side appears to be sufficient (96). 3.

POL~ANDPOLE IN VIRALANDCELLULAR DNAREPLICATION

SV40 DNA replication has been completely reconstituted with highly purified enzymes (14), and it has been shown that ~016 is required and sufficient for primer elongation on both the leading and lagging strands during SV40 replication in vitro (97). There is increasing evidence that pol S (like pol a) is part of a multiprotein complex that is responsible for a coordinated lagging-strand synthesis. First, pol 6 copurified with RF-C and pol CYin a replication-competent complex that also contained minor amounts of Fen 1 and Lig I (23; M. Stucki and U. Hiibscher, unpublished results). Second, pol S has been shown to interact genetically with the Fen 1 homolog RAD27 in budding yeast (98). Because ~016 is able to form a dimer in budding yeast via the Po132p subunit, it is interesting to speculate that a ~016 dimer that contains two catalytic subunits might be involved in coordinated leading- and lagging-strand synthesis during eukaryotic replication fork progression. Several genetic and biochemical studies suggest pol E to be the third replicative pol(66, 84, 99, IOO), whereas no essential role for human pol E in the in vitro SV40 DNA replication system was detected (94). Additional data provide strong evidence that this pol is not involved in SV40 replication (101, 102). However, there are several lines of evidence that indicate there is a function of pol E in DNA replication in eukaryotic cells (57). Interestingly, this function does not require DNA synthesis, based on work showing that the amino-terminal domain of the large pol E subunit containing both the pol and 3’ -+ 5’ exonuclease motifs is dispensable for DNA replication, DNA repair, and cell viability in budding yeast (103). Conversely, the carboxy-terminal region, lacking any known catalytic activity, is both necessary and sufficient for all of the essential functions of pol E (see below). Of course this does not mean that pol E is not participating in DNA synthesis events during chromosomal DNA replication, because there is ample evidence for this (57, 102) but it means that pol S (at least in budding yeast) is sufficient and can replace every catalytic function provided by pol E, thus representing a nice example of molecular redundancy in the cell.

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4. ROLEOFPOLEINSPHASECHECKPOINTCONTROL It has been proposed that pol E acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks (204). In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes encoding products that facilitate DNA repair. Mutant yeast cells that have a defect in pol E fail to arrest and to activate transcription of certain damage response genes. As a consequence, these cells enter into mitosis without correctly completing DNA replication. The checkpoint function of pol E has been mapped to the essential carboxy-terminal domain (104). Moreover, examination of the induction of certain genes in response to W damage revealed that Pol2p (pal E) is required to sense W damage and replication blocks when cells are in the S phase, whereas other checkpoint genes (RADS, RAD24, MEC3) are responsible for a cell cycle arrest in G, or G, (105). Finally, the S. cerevisiae gene DPBII, which has a checkpoint function, was shown to interact genetically with POL2 (106). However, the notion that such a checkpoint function by pol E is common to all eukaryotes turned out to be rather unlikely, because opposite results were reported in the fission yeast S. pombe (70). In this organism, pol E does not seem to have a role as a checkpoint sensor, in contrast to pol CY(49) (see above), suggesting that this checkpoint signal is generated prior to the elongation stage of DNA synthesis. Finally, as mentioned above, it has been shown that the essential role of the pol E carboxy terminus in budding yeast does not depend on induction of a checkpoint, which suggests that this pathway might not be essential in S. cerevisiae either (103). 5. ROLEOFPOL~ANDPOLEINEXCISIONREPAIR DNA synthesis during replication is not the only important task of ~016 and pol E (Fig. 1). The three major excision repair pathways, BER, NER, and MMR, involve a DNA synthesis step that replaces damaged or mismatched bases or nucleotides excised during repair. The BER pathway is essential in all organisms and is the main strategy to correct both spontaneous base loss or base damage and small DNA adducts. In mammalian cells, two different BER pathways have been identified. After spontaneous base loss or enzymatic removal of a damaged base by a DNA glycosylase, leaving a so-called apurinic/apyrimidinic site (AI’ site), a specific endonuclease incises the DNA double helix immediately 5’ to the deoxyribose-phosphate residue. Most probably, the main route for completion of this repair pathway is mediated by pol B in mammalian cells (107). This pol seems to be designed for this task because it contains an 8-kDa basic domain that has B-elimination activity that serves to excise the 5’-terminal deoxyribose-phosphate residue. Consequently, the same enzyme can excise and resynthesize the lesion by replacing only one

RNaseH I ?

\

1 DNA2 ?

5

3

3

5

Fen 1

F‘en 1

C RF;C

PCNA

Pol a/E

I

Fe\n 1

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277

nucleotide (the so-called short-patch BER). In budding yeast, the situation seems to be different, because a deletion of the yeast pol B homolog POL4 did not significantly result in increased sensitivity to monoalkylating agents (108). In contrast, a POL3 (~016) mutant is highly sensitive to the same treatment (109). In another yeast genetic study, pol E has been suggested to be required for BER in viva (110). However, it is also possible that alkylation damage is repaired by a different pathway (which necessitates DNA synthesis by ~016 or pol ?? ) in yeast. This is a good example of the difficulty in assigning specific pols to particular pathways using either only biochemical or only genetic approaches (57). Biochemical analysis with human and Xenopus-oocyte cell extracts or fractionated extracts of a pol B-deleted mouse fibroblast cell line indicated that BER can be completed by an alternative route when more than one nucleotide is replaced. This pathway, called long-patch BER, is strictly PCNA dependent (III-113), indicating the involvement of a PCNA-dependent pollike ~016 or pol E in the resynthesis step. Another interpretation of these data is possible based on the finding that PCNA interacts and stimulates Fen 1 (114). This enzyme is thought to be required for excision of the damaged DNA strand 5’ to the lesion (115, 116) (see below). Long-patch BER has been reconstituted with highly purified human enzymes (88, 116,117) (see Fig. 1C). It appears that pol B, ~016, and pol E are able to complete long-patch AP site repair in vitro. PCNA and Fen 1 are required for both the production and ligation of long-patch repair intermediates created by ~016 and pol E, suggesting an important role of this complex in both excision and resynthesis steps. The repair intermediates in the absence of a ligase are significantly different when in vitro assays were performed using either ~016 or pol E, which could mirror the inefficiency of pol E to perform strand displacement synthesis (88). However, the question of the physiological significance of ~016 and pol E in mammalian long-patch BER is difficult to address and remains elusive. Nevertheless, taking into account that BER is an essential repair pathway (spontaneous base loss alone occurs up to lo4 times per cell per day) and that pol B-deleted mouse fibroblast cells are viable and show normal growth characteristics and only moderate sensitivity to alkylating agents (107), we believe that long-patch BER by ~016 and

FIG. 1. Gap-filling reactions during DNA replication (A), nucleotide excision repair (B), and long-patch base excision repair (C) by DNA polymerase 8/e. Three gap-filling reactions in DNA replication and DNA repair share identical enzymes that are associated in holoenzymes for elongation and processing of the DNA synthesis patches. All of these proteins (except the singlestrand binding protein RP-A) were shown to interact directly with PCNA. (C) The apuriniw apyrimidnic (A) site is indicated. (See text and references for details.)

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pol E may be a less efficient but nevertheless important and sufficient redundancy pathway for cell viability. The NER pathway acts most effectively on bulky or helix-distorting lesions. The main function of this repair pathway in humans is the removal of W-induced DNA photoproducts caused by sunlight. Several protein factors are required to recognize the damage and remove it within an oligonucleotide of about 30 residues in length (4). The resulting gap subsequently gets resynthesized and ligated. A role of ~016 or pol E in the synthesis step of this repair pathway is consistent with the strict requirement of PCNA for NER in human cell extracts, and, indeed, several lines of evidence suggest a participation of either ~016 or pol E in the gap-filling synthesis process during NER in vitro and in viva (118). Mammalian NER was reconstituted in vitro using highly purified enzymes (119). These and other in vitro data revealed that a combination of pol E, PCNA, RF-C, RP-A, and Lig I is well suited to the task of creating NER patches. Although ~016 seems to be able to replace pol E, it gives rise to a small portion of ligated products. Addition of Fen 1 increases substantially the yield of ligated repair products, which is consistent with the ability of pol S to perform limited strand displacement synthesis (Fig. 1B).It seems, however, that pol S is unable to reach the same repair efficiency seen with pol E in vitro (86). MMR is the third essential excision repair pathway in eukaryotes. Like the other repair pathways, MMR involves two steps: damage recognition and incision, followed by resynthesis and ligation. Although the damage recognition step has been studied in some detail (6, 120), relatively little is known about the excision and resynthesis steps. There is some evidence that at least pol 6 is involved in both steps. Human cell extract fractions that are defective in supporting mismatch repair in vitro could be complemented with a partially purified fraction that contained ~016 and was free of pol (Yand pol E. Purified calf thymus ~016 also fully restored mismatch repair ability of the depleted extract, indicating that pol 6 is required for MMR in human cells. However, due to the presence of pol (Y and pol E in the depleted extract, potential involvement of one or both pols in the reaction could not be excluded (121). Another report based on genetic studies in budding yeast suggested the involvement of the 3’ + 5’ exonuclease function of ~016 and pol E in the excision step (122). Clearly, much more work remains to be done to understand in detail the enzymatic requirements and mechanisms of the excision and synthesis steps in eukaryotic MMR. 6. POLBANDPOLEINOTHERREPAIRPATHWAYS DNA lesions in the template strand cause a block to the replication machinery. Replication across such lesions occurs by a mutagenic bypass process in which a wrong base is inserted opposite the lesion, or involves

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processes that are relatively error free. Work by several groups has indicated the requirement of two pols, one for mutagenic (pal 5) and one for error-free bypass (pal I$ (9). Genetic studies implicating PCNA in this process have suggested that either ~016 or pol E may be involved as well. By the use of temperature-sensitive mutations of S. cerevisiae POL2 and POL3 it was shown that postreplicative bypass of W-damaged DNA is severely inhibited in the POL3 (pal 6) mutant but not in the POL2 (pal ?? ) mutant at the restrictive temperature. From these observations, the authors suggested a requirement of ~016 in postreplicative bypass of W-damaged DNA (123). However, a direct participation of ~016 in translesion synthesis is rather unlikely, because this enzyme is inefficient in carrying out lesion bypass synthesis in vitro (124). A study by Holmes and Haber (55) suggests pol F and pol E to be involved in double-strand break repair in vivo. MAT switching in budding yeast provides a good model for studying the involvment of enzymatic functions in DSBR via homologous recombination. Temperature-sensitive mutants of pol 6 and pol E were analyzed for their ability to complete MAT switching in vim The pol E mutant strain was almost as effective as the wild-type strain at both the permissive and the nonpermissive temperature, whereas the pol 6 mutant strain exhibited delayed and reduced MAT switching. These findings suggest that ~016 might be the major pol involved in this process, but some functional redundancy appears to exist between ~016 and pol E in DSBR as well (55).

II. DNA Polymerase Accessory Proteins A. Replication Factor C 1. GENERALDESCRIPTIONOFTHEENZYME RF-C is a heteropentameric complex composed of one large subunit (pl40/RFCl) and four smaller ones (p40/RFC4, p38/RFC5, p37lRFC2, and p36/RFC3), which interestingly all share considerable sequence similarity with each other as well as with their bacterial clamp loader counterparts in the pol III y complex (78). That all four small RF-C subunits share a common ancestor is further suggested by the fact that the genome of the archaeon Methanococcus jannaschii (125) contains only two RF-C genes, one encoding a large subunit and one encoding a small subunit homolog. The bacteriophage T4 also has a functional clamp (gp45) and a heteropentameric clamp loader composed of one gp44 and four gp62 subunits (78). Although derived from a common ancestor, the different subunits of RF-C are all essential for viability in yeast (126-129) and have been shown to have distinct functions, which involve the assembly of the RF-C complex, interaction with PCNA and DNA, and ATP hydrolysis (79,130 - 133). Some subcomplexes of

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RF-C subunits as well as the pentameric ing activity. 2. RF-CIs

complex also have PCNA unload-

ACLAMPLOADER

RF-C was first isolated by fractionation for activities that were required for SV40 replication in vitro (134). Later, by using an assay that measured stimulation of pol6 activity on primed ssDNA in the presence of PCNA, ATP, and RP-A, RF-C was also identified in yeast (135, 136) and calf thymus (82). Footprinting experiments revealed that RF-C bound DNA at templateprimer junctions in an ATP-dependent manner (137). However, RF-C on its own had little or no effect on DNA synthesis by pol CX,6, or E. An explanation for this lack of activity by RF-C on pols (Y, 6, and E is that its main function is to load the pol processivity factor PCNA onto the DNA (21, 138-140). RF-C-catalyzed PCNA loading is obligate for assembly of pol6 onto the DNA template to form the processive holoenzyme that acts during DNA synthesis of both leading and lagging strands at the replication fork (20,82,135 137, 139) (see also Section I). This is, however, by no means the only pathway requiring RF-C. As already discussed above and in more detail below, PCNA is involved in a number of DNA repair pathways, distinct from DNA replication, and because PCNA loading is required for these functions as well, RF-C is also an important component of these pathways. Two well-documented examples are NER (86,119) andlong-patch BER (88,112,113,117). Furthermore, RF-C may have separate roles in cell cycle regulation by virtue of its phosphorylation (78; A. Fotedar, personal communication) or interaction with other proteins. The yeast Rfc5p has been shown to interact with Spklp, an essential protein kinase for the transition from S phase to mitosis (141); the yeast RFC2 gene is required for an S phase checkpoint (142) and the S. pombe Rfc2p has been shown to play a key role in a DNA replication checkpoint (143). The exact mechanism of PCNA loading by RF-C is not yet understood in detail. It has been shown that RF-C dissociates from PCNA after loading it onto the DNA and does not remain directly associated with the pol 6 core (79), although the proteins can be isolated together as components of higher order complexes (144, 23). How exactly RF-C opens up the PCNA torus to load it onto the DNA is not known in detail. Studies on the homologous systems from baceriophage T4 (146-150) and E. co.& (I%), as well as the crystal structure of the 6’ subunit of the E. coli y complex (152), have given insights into how this may take place. The y complex can, on ATP binding, bind and open up the B-clamp (the structural and functional homolog of PCNA). The y/B/ATP complex then associates with the primer terminus and forms a ternary complex with the DNA. The DNA binding stimulates ATP-hydrolysis, which ejects the y complex and leaves B on the DNA (151). The mecha-

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nism of T4 clamp loading seems to be slightly different because

ATP hydrolysis is required prior to opening up the gp45 clamp (147, 149, 150). Early studies of RF-C showed that in the presence of the nonhydrolyzable ATP analog, ATP+, a stable RF-C/PCNA/DNA complex, is formed (81, 140, 153), which might implicate that the eukaryotic system more closely resembles the y complex in respect to how the PCNA clamp is loaded by RF-C (151). 3. RF-C Is LIKELY RESPONSIBLE FOR THE POLYMERASE SWITCH As discussed in Section I an important event in DNA replication is the switch from the pol (Yto ~016. An initial observation (21) suggested a role for RF-C in this mechanism. In very recent work we have shown that RF-C can displace pol (Yprior to PCNA loading and that RF-C can abrogate primer syrthesis by pol (Yat a critical length of 30 nucleotides (G. Maga, R. Mossi, and U. Hiibscher, unpublished results).

B. Proliferating

Cell Nuclear

Antigen

1. GENERAL DESCRIPTION OF THE PROTEIN Although the role of PCNA

in replication is important, it is by no means

its only known role. Indeed, as its name implies, it was first discovered as an antigen characteristically found in the nucleus of dividing cells (154, 155);

only a short while later PCNA was discovered independently by cell cycle researchers performing two-dimensional gel electrophoresis with radiolabeled cell extracts, and they named it cyclin because of the characteristic variation in synthesis during the cell cycle (156, 157). Later PCNA was shown to be an auxiliary factor for DNA replication (158-160). It can thus be argued that the role of PCNA as a cell cycle regulator was the first such discovery. The early history of PCNA research and studies of its cell cycle regulation and intracellular localization were summarized previously (161). Subsequently, PCNA has been showing up in new pathways, which may reflect the need to coordinate cellular functions such as cell cycle regulation and DNA repair with the fundamental process of DNA replication. In multicellular organisms development and differentiation add an additional level of complexity to the regulatory mechanisms required, and it seems that PCNA has a role there as well (77). PCNA is a homotrimeric ring-shaped protein with a molecular mass of 29 kDa for each monomer that occupies 120” in the ring. The crystal structure of PCNA (162, 163) revealed how PCNA can carry out its sliding clamp function on DNA by forming a trimeric ring that encircles the DNA strand, without making direct contact. In addition to its crystal structure being solved, a wealth of information about the involvement of PCNA in replication has accumulated and the list of PCNA interactors continues to grow

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rapidly, although the exact function of most of these interactions has not been clarified. Here we discuss only the best characterized functions of PCNA and must mention most of its interacting partners only briefly. 2. PCNA Is

A

CWP

THAT COMMUNICATES

WITH MANY OTHER PROTEINS The exact sites of interaction on the PCNA molecule with the three multisubunit proteins ~018, pol E, and RF-C have been studied by several groups (g&164-166); the sites have been mapped to a region on the outer front surface of PCNA involving the loop that connects the two domains of each PCNA monomer and a loop immediately preceding the C terminus (Table III). In addition, pol E has been shown to interact with a loop on the backside of PCNA (96). Th e interaction sites on the other interaction partners have not been characterized in as much detail. An interesting question involves the site of PCNA interaction in the ~016 holoenzyme, and apparently there are multiple sites of interaction (see Section I). The most intensively studied PCNA-interacting protein is the ~21”~~~’ wafl kinase inhibitor of mammalian cells (167); this kinase can inhibit DNA replication in vitro by competing for PCNA binding. Although the interaction of the two proteins has been studied in great detail by many groups, the consequences of the interaction in vivo have still not been conclusively determined. The most popular theory is that p21 can mediate cell cycle arrest in response to DNA damage by its interaction with cyclincdks but achieves an acute stop of DNA synthesis by occupying the same binding sites on PCNA as ~016. It has, however, been shown that p2 1 does not interfere with repair DNA synthesis by pol S (168). The p21 protein belongs to a family of kinase inhibitors with two identified homologs in mammalian cells (p2 7K’P1 and ~57~‘~~) as well as homologs in other species. Two of these have been shown to engage in PCNA interaction. The cyclin-dependent kinase inhibitor p57 contains a PCNA-binding site within its C-terminal region, which is necessary for p57 to suppress fully myc/RAS-mediated transformation in primary cell lines (169). The Drosophila Dacapo protein, which probably represents the insect counterpart of mammalian p21, also interacts with Drosophila PCNA (270). Another well-characterized function of PCNA is to stimulate the activity of Fen 1 (171, 172). Fen 1 has been shown to play important roles in the maturation of Okazaki fragments during replicative DNA synthesis (173, 174), in long-patch BER (115) and NER by pol 6 (86). Although Fen 1 has a potent nuclease activity in the absence of PCNA, the interaction is important for its substrate recognition and it has been demonstrated that mutation of the PCNA binding site of Fen 1 impairs its activity in long-patch BER (117, 175).

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IN DNA PROCESSES

TABLE III PROTEINS~NTERACTINGWITH

PCNA”

Protein

Presumed functional tasks

PO16

P21 Drosophila Dacapo p27 and p57 Gadd45 MyD118 MSH2 and MLHl UDG2 UNG2 XP-G MCMT CAFl YB-1

Replicative pol, gap-filling pol in nucleotide excision repair, base excision repair and postreplication mismatch repair, double-strand break repair Gap-filling pol in nucleotide excision repair, base excision repair, double-strand break repair, replication, checkpoint control Adapter for PCNA, clamp loader and possible unloader; binds to telomerase Processing of Okazaki fragments, nucleotide excision repair, base excision repair, and recombination Ligation of Okazaki fragments, nucleotide excision repair, base excision repair Control of cell cycle cdk inhibitor; up-regulated in response to DNA damage Homolog of p2 1 cdk inhibitors Unknown role; up-regulated in response to DNA damage Terminal differentiation of some cell types Mismatch repair Base excision repair Base excision repair Nucleotide excision repair Transcription Chromatin assembly factor, repair Binds cisplatin-damaged DNA

Structure Chromatin Replisome Repairsome

PCNA role Assembly of chromatin Scaffold for pols and other enzymes Scaffold for pols and other enzymes

PO1

E

RF-C Fen 1 Lig 1 Cyclins and cdk complexes

"Forreferences, see text.

Besides the roles that PCNA plays in DNA replication and BER, it has been shown to be involved in most other types of DNA repair. Reconstitution of NER in vitro with purified proteins (119) showed a requirement for RF-C and PCNA for the DNA synthesis step, and, in addition, PCNA has been shown to interact directly with XP-G, an endonuclease that functions in the incision step of the reaction (176). What the function of this interaction might be is unknown, but it could provide a physical link between the steps of incision and gap filling. An alternative possibiliy is that PCNA is engaged in recognition of some types of DNA lesions. This is suggested by the fact that YB-1, a protein that binds to cisplatin-damaged DNA, has been shown to bind to PCNA as well (177).

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Yet another type of DNA repair reaction in which PCNA has been demonstrated to play an active role is postreplicative MMR. Mutations in yeast PCNA have been shown to cause MMR defects, and direct interactions between PCNA and the yeast MMR proteins MSH2 and MLHl have also been shown (178). The exact role of PCNA in MMR is still unknown but it has been proposed to provide the signal that allows the mismatch proteins to distinguish between the parent and newly synthesized strand. In addition to these important roles in DNA repair, PCNA has been shown to interact with DNA glycosylases that are part of the normal BER machinery in mammalian cells. Both a putative cyclinlike minor uracil DNA glycosylase (UDGS) (179) and th e major nuclear uracil DNA glycosylase (UNGB) (180) h ave been shown to interact with PCNA. UNG2 localizes in replication foci during S phase and catalyzes rapid removal of dUMP residues that have been incorporated during DNA replication, probably as a part of a BER complex containing UNGB, RP-A, and PCNA that closely follows the advancing replication fork. PCNA may be responsible for guiding UNG2 to the sites of replication in the nucleus. All the DNA repair events outlined above, as well as the maturation of Okazaki fragments, must be concluded by sealing nicks in the doublestranded DNA. Mammalian cells contain four distinct ligase activities but the major replicative and repair ligase is the abundant Lig I (181). Lig I contains a consensus PCNA binding site at its N terminus and indeed binds to PCNA (182,183). As for UNGB, it has been demonstrated that PCNA binding is important for the localization of Lig I to sites of replication in the nucleus (182); additional data suggested that the replication factory targeting sequence/ PCNA binding site is required in G, to control the phosphorylationdephosphorylation status of Lig I (145). In eukaryotic cells the methylation state of the base cytosine can be inherited in a quasiepigenetic manner (184). DNA methylation is absent in Drosophila, Caenorhabditis elegans, and yeast, but in mammals DNA metbylation is involved in transcriptional regulation (185) and through that in several important regulatory processes, such as regulation of development (186) and genomic imprinting (187). Abnormal methylation can lead to several diseases, including cancer and fragile X syndrome, and it is therefore crucial that methylation patterns are correctly reproduced following DNA replication. DNA (cytosine-5)-methyltransferase (MCMT) is responsible for methylating newly replicated mammalian DNA, but how its activity is regulated is unknown. The interaction between MCMT and PCNA (188) has, however, provided some hints toward an understanding of the regulation of cytosine methylation. It was shown that a region between amino acids 163 and 174 of MCMT binds to PCNA. These residues lie inside a previously determined region that targets MCMT to sites of DNA replication (189), which is remi-

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niscent of what has been observed with UNG2 and Lig I. PCNA does not seem to alter the activity of MCMT on synthetic substrates but competition with p21 seems to affect methylation in vivo (188). One possible interpretation of these results is that inhibition by p21 could prevent methylation of damaged DNA, which would lead to mutations. Another form of epigenetic inheritance that is conserved in eukaryotic cells is mediated by the chromatin assembly factor 1 (CAF-l), a complex of three subunits, ~150, ~60, and p48 (190). In vitro CAF-1 promotes assembly of chromatin during DNA replication and in vivo it is required for inheritance of epigenetically determined chromosomal states. It has been shown that PCNA provides a signal for marking recently replicated DNA for chromatin assembly and does so through a direct interaction with the CAF-1~150 subunit (191). The role of the CAF-1 PCNA interaction does, however, not seem to be limited to postreplicative chromatin assembly, given that a mechanistic link has also been observed between DNA repair and chromatin assembly. Incubation of UV-damaged plasmid DNA in cell-free extracts revealed that de novo nucleosome assembly occurs concomitantly with NER (192, Z93), and this chromatin assembly pathway is stimulated by CAF-1 (192). As we have already discussed, PCNA is recruited to sites of DNA damage and could therefore be expected to recruit CAF-1 to these sites. That this is indeed the case has been demonstrated (J. G. Moggs and G. Almouzni, personal communication). The PCNA interactions that we have discussed so far mostly involve PCNA bound to DNA, but in replicating cells PCNA continuously cycles between a chromatin-bound detergent-insoluble form in S phase and a soluble form when DNA replication is not taking place. A large population of unbound PCNA thus also exists and some of this PCNA has been found to associate in complexes with p2 1 and several pairs of cyclincdks, including cyclin D/cdk4, cyclin E/cdk2, cyclin A/cdk2, and cyclin B/cdc2 (194). It seems almost certain that these interactions play a role in coordinating the cell cycle with DNA replication and repair, but their exact role is still relatively poorly understood. On one hand it may be that PCNA can attract some of its interaction partners to active cyclincdk complexes for being phosphorylated; on the other hand it has been suggested that excessive levels of cyclin Dl can repress cell proliferation by inhibiting DNA replication and cdk2 activity through binding to PCNA and cdc2 (195). We have shown a direct interaction between PCNA and Cdk2 that involves the C-terminal part of PCNA and the catalytic site of Cdk2. Binding of PCNA to Cdk2 resulted in an inhibition of the kinase activity and in inhibition of pol 6 in a PCNA-dependent assay. PCNA and Cdk2 form a complex in S phase. Based on our results we propose that PCNA brings Cdk2 to proteins involved in DNA replication and can act as a

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“stirrup” for cyclin A/cdk2 to further target proteins involved in DNA transactions (S. Koundrioukoff and U. Hiibscher, unpublished results). The list of reported PCNA interactors presented here is by no means complete and the collection keeps on growing. It should, however, be evident that PCNA has more than a dual role in DNA transactions; the paradigm could sound more like “PCNA has a role in every cellular pathway that involves DNA.” The notable exception whereby a role for PCNA has not been demonstrated is RNA transcription. Although the PCNA homolog of the phage T4, the gp45 protein, has been shown to play an important role in transcription of genes that are expressed late in the viral life cycle (196), and other examples of the involvement of viral PCNA homologs in transcription exist (197, 198), eukaryotic PCNA has not been shown to be directly involved in tran scription. This possibility has, however, not been formally excluded either.

III. Transcription Factors and Their Role in Activation of DNA Replication A.

Intrpductory

Remarks

An important parameter that determines the order of chromosome replication is transcriptional activity: most, but not all actively transcribed genes are replicated early in the S phase, whereas quiescent genes are replicated at later times (199). This is true even when two copies of the same gene reside in the same cell and one is transcriptionally active while the other is inactive (200, 201). Due to this observation it was proposed that the transcription process may play an important role in determining the order of replication of genes during S phase. We know that transcription by RNA polymerase II (pal II) in eukaryotes is carried out with the aid of many accessory proteins, including the general transcription factors (GTFs) TFIIA, -B, -D, -E, -F, and -H and the components of the MediatorSRB complex, which interacts with the C-terminal domain (CTD) of the pol II major subunit (202, 203). Transcription factors form a large family of regulatory proteins that can positively or negatively influence transcription by binding to regulatory elements in promoter elements. In the past 8 years, it has become clear that many transcription factors are multifunctional and also influence DNA replication. B. Lessons from Viral Systems Most of our knowledge about the role of transcription factors in activating DNA replication comes from studies of DNA tumor viruses (204-206). Replication origins are classified and viral origins belong to so-called simple origins, together with those origins found in mammalian (human, mouse) mitochondria, in yeasts (S. cerevisiae and S. pornbe), and in slime mould (207).

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A typical viral origin is from 50 to about 1000 bp long and is defined by cis-acting mutations that prevent DNA replication. It consists of at least four elements, three of which [origin recognition element (ORE), DNA unwinding element (DUE), and A/T-rich element] are essential and therefore referred to as the origin core. The fourth element contains transcription factor binding sites (aux-1 and aux-2) that flank one or both sides of origin core (199, 207). Aux-1 is p roximal to the DUE element, and aux-2 is proximal to the A/T element. Origin auxiliary components stimulate replication only when they bind one or more transcription factors, and only when the transcription factor contains an activation domain that specifically interacts with the replication machinery (199,207,208). In some viral genomes, such as SV40, polyomavirus and Epstein-Barr virus genomes, the same sequence elements that function as promoters or enhancers in transcription also function as auxiliary components in replication. Substitution of the polyomavirus enhancer with the immunoglobulin gene enhancer conferred tissue-specific replicator-y ability to the virus, which showed for the first time the importance of these transcriptional elements in regulating viral DNA replication (204). In addition, the auxiliary sequences of the SV40 and polyoma virus origins of replication located adjacent to the binding site for the large T antigen contain elements recognized by cellular transcription factors such as Spl, APL and ~53. These transcription factors can increase viral origin activity up to lOOO-fold (204, 206, 209). Heterologous transcription factors can also activate viral replication when tethered to the origin of replication. For example, factors such as NF-KB, VP16, ElA, bovine polyomavirus E2, and yeast Gal4 stimulate polyomavirus DNA replication (209-211). H ow do transcriptional elements regulate viral origin activity? First, transcription factors could regulate the temporal order of DNA replication during S phase, just as they initiate transcription of different genes at different times during the cell division cycle (212). Second, the ability of a particular transcription factor to stimulate a particular origin may be limited to specific members of a transcription factor family, and to the availability of specific coactivator proteins (199,212, 213). Due to the lack of space, we refer the reader to several reviews discussing the role of transcriptin factors in viral DNA replication (208, 214, 215).

C. Lessons from Yeast Compared with the experiments in viral systems, the role of transcription factors in cellular DNA replication is less well understood. Nevertheless, analyses of eukaryotic cellular origins of DNA replication have been greatly facilitated by the biochemically and genetically tractable yeast S. cerevisiae.

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DNA sequences of S. cerevisiae were identified that enabled the extrachromosomal replication of plasmids (216). These sequences are termed autonomously replicating sequences (ARSs) and they act as genuine origins of replication (217, 218). A detailed mutational analysis of one origin, ARSl, has led to identification of two essential elements, A and B (213). Element A contains an 11-bp consensus sequence that is conserved among all origins in S. cerevisiae. It is the binding site for the initiator protein called origin recognition complex (ORC) (219). The B element is composed of three functional sequences: Bl, B2, and B3, which are collectively essential for origin function (213). The Bl element is important for ORC binding and additional functions in replication initiation (220). The function of the B2 element is not clear. The B3 element contains a binding site for the yeast protein Abfl that functions as a transcriptional activator and repressor protein (221, 222). It was shown that, like enhancers in viral replication, the function of the B3 element of ARSl in plasmid replication can be replaced by the binding site for other yeast transcription factors such as Gal4 and RAPl. The ability of Gal4 and RAP1 to activate DNA replication resides in their transacting transcriptional activation domains (213). In addition, others have analyzed which type of transcriptional activation domain can activate a yeast cellular origin of replication in a chromosomal context (223). It has been reported before that acidic trans-activation domains derived from herpes simplex virus VP16 and from the tumor suppressor ~53 can stimulate viral DNA replication (224-226). Acidic activation domains can also activate a cellular origin of replication in a chromosomal context: when tethered to the yeast ARSl origin of replication, both VP16 and p53 acidic activation domains enhanced origin function (223). In addition, the C-terminal acidic region of the ABFl was sufficient for activating ARSl function when tethered to the origin. These findings strongly suggested functional conservation of the mechanisms used by the acidic activation domains to activate viral DNA replication in mammalian cells and chromosomal replication in yeast. Moreover, these experiments showed that activation of DNA replication and transcription by acidic activation domains has many common characteristics. A 50-amino acid C-terminal region of the Abflp seems to be sufficient to stimulate the function of ARSl21 origin when tethered to it. When tested for transcriptional activation of a EacZ reported gene, the same 50-amino acid C-terminal region of the Abflp had negligible transcriptional activation potential, suggesting that activation of DNA replication at ARS12 1 may occur independently of a transcriptional activation domain (227). Unlike ARSl, activation of ARSl21 by ABFl cannot be replaced by other replication factors, suggesting a special function of Abflp at ARS12 1.

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What is the exact mechanism by which transcription factors activate cellular DNA replication in viva? Results suggest that chromatin remodeling might be an important pathway utilized by transcription factors to activate yeast chromosomal replication (228). This extends and substantiates previous in vitro studies of the transcription factor’s role in activation of viral DNA replication (229,230). When tethered to a cellular replication origin, a fusion between the Gal4 DNA-binding domain and the C-terminal region of the breast cancer protein BRCAl, which has been previously shown to activate transcription in yeast as well as in mammalian cells, alters the local chromatin structure and stimulates chromosomal DNA replication. Cancer-predisposing mutations in BRCAl that abolish transcriptional activation function also prevented chromatin remodeling and activation of DNA replication (228). BRCAl is implicated in transcription (231,232), repair (233), replication, and recombination (234, 235). BRCAl is associated with chromatin-modified proteins such as BRCAB (236), ~300, and hBRG1 (237). In addition, BRCAl also interacts with components of transcription and repair machineries, such as mammalian pol II holoenzyme (238) and RAD51 (234). Although replication assays showed that the BRCAl activation domain is capable of activating an ARSl origin of replication in yeast, a direct involvement of BRCAl in mammalian DNA replication has to be established. Additional results suggested that DNA replication in yeast can be activated by recruitment of the pol II transcription complex (239). A defined, single interaction between a DNA-bound derivative of the activator Gal4 and GalllP, a mutant form of the pol II holoenzyme component Galll, suffices for stimulating DNA replication, as it does for transcription. Moreover, our results showed that recruitment of TATA-binding protein (TBP), which can activate transcription from a gene promoter, also stimulates DNA replication from ARSl origin (239). Th us, a DNA-binding protein does not need to interact directly with replication factors in order to activate replication from ARSl. In analogy with the implications of the Ga14-GalllP interaction in gene activation, stimulation of replication in these experiments does not require direct interaction of the DNA-binding protein with the machinery that helps in removing inhibitory chromatin structures (240). This model is also consistent with work (241) in which it was shown in Xenopus oocytes and HeLa cells that MCM proteins copurify with the pol II holoenzyme complex and GTFs. In addition, antibodies against MCM2 specifically inhibited transcription by pol II in microinjected Xenopus oocytes. The MCM 2-7 proteins, a family of six conserved proteins, are essential for the initiation of DNA synthesis at replication origins in S. cerevisiae (242, 243). Each of these proteins has a corresponding homolog in all eukaryotes examined so far (244), suggesting that their role in DNA replication initiation is universal.

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Studies in S. cerevisiae indicate that mutations in the MCM 2-7 genes prevent initiation of DNA synthesis at replication origins in plasmids and chromosomes (245,246). Transactivators could recruit MCM proteins to origins of replication via contacts with pol II holoenzyme complex and thereby stimulate DNA replication (241). Furthermore, it could well be that MCM proteins play a previously unsuspected role in transcription. This hypothesis is in agreement with observations of a correlation between transcriptional activation by Statla and its ability to bind MCM5 (247). Taken together, all of these results raise the possibility that natural transcriptional activators binding near replication origins also activate replication by recruitment of the pol II transcription complex through direct interactions with one or more of its components (239, 241).

D. How Might DNA

Transcription

Factors Help in Activating

Replication?

The important and direct role of transcription factors in the initiation of DNA replication has been well substantiated through several experiments performed with animal viruses and S. cerevisue (208, 218, 228, 230). It has been postulated that involvement of the same transcription factors in the activation of DNA replication from origin sites as well as transcription from gene promoters requires that these proteins have either two different biochemical activities or one single activity that stimulates two distinct processes (213). That some transcriptional activators might indeed be endowed with different biochemical activities has been indicated by results of in vitro experiments showing that these proteins can interact not only with components of the pol II transcription complex but also with replication factors, e.g., the activation domains of VP16 and p53 can interact with RP-A, an essential component of the replication machinery (225,248,249). Therefore, these results indicate that transcriptional activators might stimulate DNA replication from cellular origins by recruitment of replication factors to origin sites, i.e., the same mechanism by which some activators have been shown to stimulate viral DNA replication (208). However, results of in vivo experiments showed that replication from a cellular origin site (ARSl) in yeast can be stimulated by the same biochemical activity (proteinprotein interaction) of a DNA-binding protein that also activates transcription from a gene promoter (239). Because this molecular interaction has been shown to activate transcription by recruiting the pol II transcription complex to DNA (250), th ese results strongly suggested that the same mechanism, i.e., recruitment of the pol II transcription complex, applies to this instance of activation of DNA replication. These results more broadly raise the possibility that natural transcriptional activators binding near replication origins also activate replication by

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recruitment of the pol II transcription complex through direct interactions with one or more of its components. The recruited transcription complex may cause stimulation of DNA replication in a number of ways. One attractive possibility is that recruitment of this complex by transcriptional activators modifies chromatin structures to facilitate formation of the replication complex at the origin site. In fact, it has been demonstrated that acid activators can counteract the nucleosomal repression of transcription and that the antirepression function may be mediated by several chromatin remodeling systems (240, 251). These data strongly suggest that chromatin reconfiguration might be a highly conserved feature of transcription factors in activation of eukaryotic replication as well as transcription. In this respect it will be important to determine which nuclear machinery a transcription factor has to stimulate at a particular time and location. This may include the chromosomal region with which the transcription factor is associated (replication origin versus transcriptional promoter), the physiological state of the cell (proliferating versus nonproliferating), and specific association of transcription factors with highly condensed chromatin. In this way, origin sequences simply may not be accessible to transcription factors in highly condensed regions of the genome. In conclusion, it is now clear that there are several mechanisms by which transcription factors can facilitate initiation of DNA replication in eukaryotic cells. The mechanisms are not mutually exclusive, and might be, as in the case of different gene activation mechanisms, origin specific. The findings in yeast are highly interesting, and future studies should prove to be extraordinarily exciting for understanding the regulation of DNA synthesis by transcription factors.

IV. Perspectives and Conclusions We have exemplified that nature has evolved complex intertwined proteins that can maintain the integrity of the genome. Our simple-minded view that a particular enzyme machinery is responsible for a certain event has to be revised completely. We do not know, however, why, when, and how a particular enzyme protein machinery is called to a particular DNA transaction. The few selected examples, pols CL,6, and E, RF-C, PCNA, Ga14, ~53, BRCAl, and ABFl, clearly indicate that proteins/enzymes are multifunctional and that their different tasks must be coordinated depending on the physiological state of a cell or an organism and its response to environmental disturbances (e.g., DNA damage). The most fascinating versatile example among them is PCNA, which can interact with almost all important protein families involved in DNA metabolism and thus can act as a coordinator (Table III), a

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stirrup, or a bridge in DNA replication, in several DNA repair events, in cell cycle control, in checkpoint control, and possibly even in transcription. We have now to learn in great detail how all these enzymes and proteins can interact and what the triggers are that engage a particular enzyme in a particular event. The combined efforts of biochemical, genetic, and cell biological approaches will be required to obtain a complete understanding of these most essential events in life. Last but not least, important as-yet unsolved medical problems such as cancer, aging, and other genetic diseases will profit from this basic understanding of how enzymatic machineries can maintain the software of the cell with an integrity that guarantees life, from unicellular organisms to humans.

ACKNOWLEDGMENTS We thank A. Barberis for exciting suggestions. Y.-S. Seo and A. Fotedar are acknowledged for communicating unpublished observations. The work in the authors’ lab has been supported by grants from the Swiss National Science Foundation to ZOJ and UH, the Swiss Cancer League to MS and UH, the EU-TMR project ERBMRXCT CT970125 to IS and UH, and the Kanton of Ziirich to IS, ZOJ, and UH.

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