Mechanisms and consequences of replication fork arrest

Biochimie 82 (2000) 5−17 © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400003448/REV

Review

Mechanisms and consequences of replication fork arrest Olivier Hyrien* Génétique Moléculaire, École Normale Supérieure, 46, rue d’Ulm, 75230 Paris cedex 05, France (Received 20 July 1999; accepted 8 September 1999) Abstract — Chromosome replication is not a uniform and continuous process. Replication forks can be slowed down or arrested by DNA secondary structures, specific protein-DNA complexes, specific DNA-RNA hybrids, or interactions between the replication and transcription machineries. Replication arrest has important implications for the topology of replication intermediates and can trigger homologous and illegitimate recombination. Thus, replication arrest may be a key factor in genome instability. Several examples of these phenomena are reviewed here. © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS replication fork barrier / replication fork pausing site / replication termination / recombination / genome instability

1. Introduction DNA replication consists of three steps: initiation, elongation, and termination. Replication forks usually proceed at a constant speed along the DNA (about 1000 bp/s in prokaryotes, and 10–50 bp/s in eukaryotes). However, replication forks can also be stalled transiently at replication fork pausing sites (RFPs) or irreversibly at replication fork barriers (RFBs). DNA downstream of a RFB has to be replicated by a converging fork that ultimately merges with the arrested fork. Therefore RFBs are sites of replication termination. In prokaryotes, termination usually involves arrest of replication forks at specific sequences called replication termini [1-4]. Replication termini are binding sites for specific replication terminator proteins, and the terminiterminator protein complexes act as polar RFBs. The arrested fork then serves as an arrest site for the second, converging fork. In eukaryotes, sequence-specific termination seems to be the exception rather than the rule. Eukaryotic DNA replication initiates at numerous replication origins, and most termination events probably occur wherever converging forks happen to meet, i.e., within zones that can span several kb [5, 6]. Thus, eukaryotic replication forks must possess an intrinsic ability to stop progression and to disassemble the replication machinery when they meet. This suggests that specific termination sequences are not necessary for replication termination and may serve other purposes. Experimental evidence in both prokaryotes and eukaryotes suggests that arrest of replication forks is associated with recombination [2, 7]. We review here the * Correspondence and reprints

various mechanisms of replication fork arrest, the topology of arrested replication intermediates (RIs), and recent progress on the mechanisms of recombination at arrested forks. 2. Mechanisms of replication fork arrest Replication elongation in vivo can be arrested by various kinds of obstacles: the structure of the DNA template itself, lesions on DNA, specific protein-DNA complexes, RNA-DNA hybrids, and collisions with the transcription machinery. Evidence linking in vivo replication arrest to in vitro inhibition of either helicases and polymerases, two classes of key enzymes at the fork, has been reported. A summary of various instances of replication arrest is presented in table I. 3. Arrest of replication by specific DNA structures DNA polymerization in vitro is inhibited by a variety of DNA sequences such as inverted repeats, polypurine stretches, or some microsatellite DNA sequences, which can form stable secondary structures such as hairpins, triplexes and quadruplexes when subjected to negative DNA supercoiling [2, 8]. As detailed below, some of these repeats were found to inhibit DNA replication in vivo. 3.1. Arrest of polyomavirus replication by (dG-dA)n.(dT-dC)n microsatellites Mitomycin-C can induce onionskin replication of mammalian chromosomal DNA flanking the replication origin of an integrated polyomavirus DNA. In a study of

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Table I. Summary of various instances of replication arrest. The list is not limitative. Type of arresting structure

Organism

Polarity

DNA structure GAn Polyomavirus

Secondary structure (in vitro) triplex

SV40

CGGn

E. coli

Klenow

Antihelicase activity (in vitro)

Transcription arrest

SV40 Tag

polar

hairpin (both) quadruplex (CGGn)

Association with recombination Putative role in viral DNA amplification Instability possibly due to replication slippage

Calf thymus DNA pol α

CAGn

Remarks

Pausing not demonstrated in human diseases

Klenow, T7 pol Human DNA pol β

Protein-DNA complexes TusE. coli Ter RTP-τ B. subtilis

polar

RTP-ψ

B. subtilis

polar

EBV FREBNA-1 CENCBP rDNA RFB?*

EBV

bipolar

S. cerevisiae

bipolar

S. cerevisiae

polar

Human

bipolar

Mouse

polar

RNA-DNA hybrid Opposing E. coli ColE1 origins

polar

Transcription tRNA S. cerevisiae genes

polar

rDNA RFBTTF-I

Arrest of DNA polymerases (in vitro)

polar

DnaB, SV40 Tag DnaB, SV40 Tag

isopolar

homologous and illegitimate

Abolished by transcription in permissive direction Abolished by transcription in permissive direction Induced by stringent response; reversible Weak arrest in absence of EBNA-1

ERC excision

Arrest independent of transcription

isopolar

DnaB, SV40 Tag

antipolar

ERC excision linked to aging Arrest reconstituted in vitro with purified TTF-I Developmental regulation in frogs

DnaB

Knotting of arrested bubbles

Pausing zone (500bp) larger than a tRNA gene

Hyrien

*Putative protein-DNA complex. In contrast to the binding of TTF-I to human and mouse rDNA, no binding of a specific protein to the S. cerevisiae rDNA RFB has been demonstrated to date, although Fob1 is a plausible candidate.

Mechanisms and consequences of replication fork arrest

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this phenomenon, it was observed that a region containing the repeat (dG-dA)n.(dT-dC)n could slow down replication fork movement [9, 10]. This simple repeat sequence, when cloned into SV40 DNA, slows down DNA replication fork movement in vivo, as assessed by two-dimensional (2D) gel electrophoresis of replication intermediates (RIs) [11]. In vitro, this sequence can adopt triplex structures, and inhibits both DNA synthesis by purified DNA polymerases [12, 13] and DNA unwinding by the SV40 T antigen helicase [14]. Whether inhibition of a helicase or a polymerase is the actual mechanism of replication arrest in vivo remains to be determined. 3.2. Arrest of replication forks by trinucleotide repeats in E. coli More than a dozen human disorders are attributed to the expansion of simple DNA repeats within different human genes [15, 16]. Trinucleotide repeats, particularly CGGn and CAGn, account for most of these cases. The probability of expansion strongly increases with repeat length, suggesting the involvement of some unusual DNA structure. The mechanism of repeat expansion is unknown, but the observed polarity of expansion and other data implicate replication in this process. The progression of replication forks through these repeats cloned into a bacterial plasmid was followed using 2D gel electrophoresis of RIs [17]. These repeats stall the bacterial replication fork in vivo. This stalling depends on the length of the repeats and their orientation relative to the replication origin. Replication arrest is unlikely to be mediated by tight protein binding to repeated DNA, since inhibition of protein synthesis by chloramphenicol enhances, rather than abolishes, replication blockage. Formation of unusual DNA structures in the lagging-strand template may account for the replication blockage, and may have relevance to repeat expansions in humans.

Figure 1. Organization of the E. coli chromosome for replication (adapted from [1]). The origin of bidirectional replication, oriC, the movement of the two replication forks (dotted lines), and the termination sites (Ter) are indicated. The circles denote Tus monomers. The T shape of the Ter sites denotes their polarity; replication forks are arrested when they meet the flat side of the T. The enlargement of the termination region shows where the first arriving fork (either the clockwise or the counterclockwise fork) is stopped. The second arriving fork then stops when it meets the first arrested fork.

4. Termination of bacterial chromosome replication The best studied example of replication arrest caused by specific proteins bound to specific DNA sequences is the termination of bacterial replication (reviewed in [14]). 4.1. Replication termini Replication termini were first discovered in the plasmid R6K [18, 19] and subsequently in the E. coli [20, 21] and B. subtilis [22-24] chromosomes. In both species, chromosomal replication initiates from a single origin. Two replication forks travel in opposite directions around the circular chromosome and terminate in a region 180° away from the origin (figure 1). The termination region is flanked in both species by six polar RFBs that are

arranged as two inversely oriented sets so as to form a replication trap. The first approaching fork can pass through the first three RFBs it encounters, but is blocked at the first oppositely oriented RFB. The other, converging fork then meets the arrested fork and replication is terminated without reduplication of the sequences between the two sets of RFBs. The two sets of RFBs are separated by only 59 bp in B. subtilis. In E. coli they are separated by 270 kb, so that the two converging forks may sometimes meet within the replication trap rather than at the termini. It is unclear why each chromosome has several termini rather than the minimal requirement of two. Additional termini may act as a fail-safe mechanism to block forks that pass the first arrest site.

8 4.2. Properties of termini-terminator protein complexes Neither the sites responsible for termination nor the replication terminator proteins in the two bacteria are related to each other. E coli Ter sites are 23 bp long sequences that recognize and bind a monomer of a 36 kDa protein named Tus (for terminus utilization substance, also called Tau) [25, 26]. The Ter-Tus complex is very specific and long-lived (Kd = 3.4 × 10–13 M; half-life of 550 min for the Tus-TerB complex) [27]. The analogous sites in B. subtilis (named Ter or τ) are 47 bp long imperfect inverted repeats, each consisting of a core sequence and an auxiliary sequence, bound by two dimers of the replication terminator protein (RTP). RTP has a monomer molecular mass of 14.5 kDa and forms a stable dimer. The core sequence binds a dimer of RTP, and this protein-DNA complex allows the binding of a second dimer to the auxiliary site [28, 29]. The auxiliary sequence does not bind RTP in the absence of a core site. The interaction of two dimers is absolutely required for replication arrest [29, 30]. In vitro, Ter-Tus complexes stall reconstituted replication forks initiated at either oriC [31] or ColE1-type origins [32]. There is no in vitro replication system currently available in B. subtilis. However, the RTP-τ complex of B. subtilis has been shown to block replication fork of E. coli both in vivo and in vitro [33, 34]. Both Tus and RTP have been shown to inhibit the unwinding reaction catalyzed by purified E. coli replicative helicase DnaB in an orientation-dependent manner, when assayed on partially duplex substrate contains a Ter site placed in the proper orientation [31, 35]. Several (but not all) other helicases, notably the SV40 T antigen, are also impeded by Tus-Ter complexes [36]. Interestingly, both Ter-Tus and Ter-RTP complexes are also able to arrest several RNA polymerases with the same polarity as replication forks [37, 38]. When transcription is allowed to invade a replication terminus from the permissive direction, the terminator protein is displaced from the terminus DNA and arrest of replication fork is abrogated. Thus, opposing transcription could potentially regulate replication arrest at termini. The crystal structures of RTP and of the Ter-Tus complex have been determined. The RTP protein belongs to the winged helix family of proteins, and its closest structural homologue is histone H5 [39]. The DNAbinding, dimer-dimer interaction, and DnaB interaction domains of RTP have been determined in combination with mutagenesis and biochemical studies. In particular, mutations at Glu-30 and Tyr-33 located within a hydrophobic patch cause partial and complete loss of contrahelicase activity without impairing the DNA binding activity, dimer-dimer interaction, or dimerization properties of the protein [39, 40]. This indicates that the simple clamping of the RTP protein on the DNA is not sufficient to provoke replication arrest. Replication arrest requires

Hyrien specific interaction between RTP and the replicative helicase. The Tus protein is structurally unrelated to other known protein structures, most notably RTP [41]. The Tus protein has not been analyzed by mutagenesis as thoroughly as the RTP protein. Most of the mutants that abolish contra-helicase activity localize within the DNA binding domain. 4.3. Role of replication termination systems The function of replication arrest at termini is not obvious. Termination by a Tus-mediated mechanism is not required for E. coli viability because tus can be deleted without affecting growth. Brewer [42] has suggested that the termini prevent replication forks from replicating the chromosome in a direction opposite to transcription. In the E. coli chromosome, more than 90% actively transcribed genes are oriented with their 5’-end toward the origin so that replication and transcription are codirectional, while weakly transcribed genes are oriented at random. Head-on collisions between replication and transcription thus appear to be specifically avoided. There is evidence that both head-on and codirectional collisions disrupt ongoing transcription, but that only head-on collisions cause slowing down of replication forks [43]. Plasmids carrying oriC and two Ter sites oriented as they are on the E. coli chromosome have been used in replication reactions reconstituted with purified proteins to demonstrate that Tus prevents overreplication in vitro [44]. In the absence of Tus, the DnaB helicase frequently fails to stop when a round of bidirectional replication is complete. As a result, DnaB helicase continues to unwind DNA, attacking the duplex between one of the original template strands and a newly synthesized strand, which leads to the generation of long DNA multimers. The contrahelicase activity of the Ter-Tus complex prevents this runaway replication in vitro. Evidence for a similar role of the Ter-Tus complex in plasmid replication in vivo has been reported [45]. 4.4. Regulation of replication fork pausing: the B. subtilis replication checkpoint In B. subtilis, induction of the stringent response results in the arrest of chromosomal replication in two regions that are located 200 kb on either side of the origin of replication [46, 47]. This replication block, which has been referred to as a replication checkpoint [48], is correlated with high levels of the alarmone ppGpp, depends upon RelA (ppGpp synthetase I), and involves the RTP protein normally active at the chromosomal terminus. When the stringent response is relieved, replication resumes close to or at the blocked sites. There appear to be several stop sites within each stringent terminator (STer) region. One of these sites has been characterized in

Mechanisms and consequences of replication fork arrest detail [49]. It contains a 17 bp sequence sharing 76% identity with the core sequence of a normal chromosomal terminus. This sequence is essential for stringent arrest in vivo. It binds a single RTP dimer in vitro, with a Kd similar to that reported previously for normal termini. Analysis of an RTP mutation indicates that interaction between two RTP dimers may not be required for arrest at STer sites, in contrast to the mechanism operating at the normal terminus. This difference is not unexpected if inappropriate arrest at STer sites is to be avoided during normal replication. In E. coli, the stringent response causes replication arrest at the level of initiation at oriC and not at other sites [50]. 5. Replication arrest by specific DNA-protein complexes in eukaryotic chromosomes 5.1. The replication fork barrier in the Epstein-Barr virus (EBV) genome The Epstein-Barr virus (EBV) latent origin of replication, oriP, contains both the initiation and termination sites of DNA replication [51]. It is composed of two essential elements separated by approximately 1 kb, the family of repeats (FR) and the dyad symmetry (DS) element. The FR contains 20 tandemly repeated copies of a 30 bp sequence, each containing a binding site for EBNA-1, the only viral protein required for latent replication. The FR is important for nuclear retention of the viral genome. The 65 bp DS element contains four binding sites for EBNA-1. Replication of an oriP plasmid initiates within or near the DS region. The leftward-moving fork is arrested within the FR; the other fork travels around the plasmid until it merges with the arrested fork. In intact EBV genomes (≈ 170 kb), replication forks also stall at the FR but at a variable level among different cell lines [52]. They also stall at two small, highly expressed genes located 0.5 to 1 kb upstream of oriP and transcribed by RNA polymerase III in the same direction as replication. The mechanism of replication arrest at the FR was studied in an HeLa cell extract using SV40 origin-based plasmids containing a variable number of the FR repeats [53]. In the presence of purified recombinant EBNA-1, termination occurs at the repeats as observed in vivo. Reducing the number of repeats from 20 to 6 has little effect, but pausing is not detected with two repeats. The substitution of three tandem copies of the DS for the FR also results in pausing, suggesting that multiple EBNA-1 binding sites are important irrespective of spacer sequences. Investigation of a series of truncated EBNA-1 proteins has shown that the complete DNA binding and dimerization region of EBNA-1 (amino acids 459–607) is sufficient to elicit the pausing of replication forks, that deletion

9 of amino acids 459–470 abrogates pausing, and that the phosphorylation status of the protein is not important [54]. Crystal structure of the EBNA-1 DNA binding and dimerization domains bound to DNA shows that amino acids 461–470 form an extended chain that makes extensive contacts with the minor groove of the DNA binding site [55]. Loss of these contacts results in a decreased affinity. Finally, EBNA-1 bound to the FR inhibits DNA unwinding in vitro by the SV40 T antigen and the E. coli DnaB helicase in an orientation-independent manner [54]. It should be noted that in the absence of EBNA-1, forks also pause at the FR but to a much lower degree. This may be due to some unusual secondary structure or to the binding of some protein(s) present in the HeLa cytosol. In summary, the mechanism of fork pausing at the EBV FR is reminiscent of that operating at prokaryotic termini, since it requires the binding of a specific protein with antihelicase activity. However, it is less efficient and not polar. Furthermore, replication fork pausing is not detected in vitro in the presence of a truncated EBNA-1 protein that retains antihelicase activity. Further work is required to investigate the relationship between fork pausing and antihelicase activity. 5.2. Replication fork pausing at S. cerevisiae centromeric DNA sequences A transient arrest of replication forks occurs at the centromeres of chromosomes I, III, and IV, and probably at all centromeres of S. cerevisiae [56]. Pausing occurs when forks approach the centromere from either direction and is relatively brief, lasting for an estimated 0.1– 0.2 min. Termination signals are not detected at the centromeres. Point mutations in CEN3 that abolish the binding of centromere binding protein abolish replication fork pausing. The ability to pause replication forks correlates with the ability to form a nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. It is not clear whether the centromeric protein-DNA complex is disrupted to allow the fork to move through. 5.3. The replication fork barrier in eukaryotic ribosomal genes The best characterized eukaryotic replication termini are those found in the genes coding for the ribosomal RNAs (rDNA) (reviewed in [57]). In most eukaryotes, the rDNA is organized as a few hundred tandem repeats, each of which includes a transcription unit (TU) for the large rRNA precursor and an intergenic spacer (IGS) that contains the regulatory elements for rRNA transcription. In general, replication initiates within the IGS. A prominent and conserved feature of rDNA replication is the presence of a RFB in the IGS near the 3’ end of the rRNA gene, in close proximity to the rRNA transcription termination site.

10 In S. cerevisiae, where this phenomenon was discovered, the RFB is polar and efficient, arresting completely and selectively forks that move in the direction opposite to transcription [58-60]. Consequently, replication of the rDNA is essentially unidirectional. It was suggested that the RFB evolved to prevent collision between replication and transcription complexes [60]. Interestingly, whereas transcriptionally active and inactive rRNA genes coexist in yeast cells, forks arrested at the RFBs are only found downstream of active genes [61]. Because of the RFB polarity and location near the transcription terminator, it was suggested that transcription itself might be responsible for the arrest of replication forks. However, genetic experiments have shown that the fork arrest occurs independently of the act of transcription [60, 62]. First, the RFB persists in a strain containing a disruption of a catalytic subunit (RPA135) of RNA polymerase I. Second, the RFB persists when transplanted on a plasmid not involved in rRNA transcription. Third, transcription by RNA polymerase II of a plasmid copy of the 35S transcription unit lacking the RFB does not generate a barrier. It was suggested that the binding of one or more proteins to the RFB is responsible for polar fork arrest, similar to the mechanism operating at E. coli and B. subtilis replication termini. Two types of proteins have been involved in replication arrest in the rDNA. First, the S. cerevisiae RFB overlaps the E element of HOT1, a recombinational hotspot in yeast rDNA [63, 64]. Genetic analysis identified the FOB1 gene, whose mutation causes a simultaneous loss of both HOT1 and RFB functions [65]. The FOB1 gene has no homology with any other known gene. Second, the rRNA transcription termination factor TTF-I has been implicated in replication arrest at the RFB in mouse and human cells. As in many other species, mouse RFBs are polar [66]. However, human RFBs appear to arrest forks in both directions [67]. The RFBs in human and mouse cells both consist of several closely spaced arrest sites that colocalize with the rRNA transcriptional terminator elements known as the SalI boxes. These elements contain binding sites for the RNA polymerase I transcription termination factor, TTF-I. Importantly, replication arrest can be reconstituted in vitro in HeLa cell extracts using mouse rDNA cloned in an SV-40 replication origin-based vector [68]. In this system, replication arrest is polar and occurs at a single site, 28 bp downstream of Sal box 2. Mutagenesis and immunodepletion experiments show that: i) TTF-I is necessary for replication arrest in vitro; ii) arrest requires a domain of TTF-I that is also required for transcription termination; iii) in addition to the TTF-I binding site, a GC-rich stretch is required downstream of the SalI box; and iv) a T-rich stretch further downstream enhances replication arrest. The arrest at multiple sites observed in vivo could depend on some factors not present in the HeLa cell extract. It should be noted that the yeast equivalent of TTF-I, Reb1, has not yet been implicated in

Hyrien RFB activity. Furthermore, no DNA binding activity has been reported for the yeast Fob1 protein, although it is clearly required for replication arrest at the yeast RFB. So far nothing excludes that Fob1 may actually regulate the activity of a yet unidentified DNA binding protein. In contrast to the bacterial terminator proteins, which stop replication and transcription progressing in the same direction, TTF-I blocks replication and transcription in opposite directions. Interestingly, TTF-I can also function to remodel nucleosomes over the mouse ribosomal gene promoter, thus removing chromatin-mediated repression of rDNA transcription [69, 70]. This novel activity necessitates a domain of TTF-I that is required for both transcription termination and RFB activity. So far, no contrahelicase activity has been attributed to the TTF-I protein.

5.4. Developmental regulation of RFPs and RFBs in the rDNA

In early Xenopus embryos, where the rRNA genes are not transcribed, replication forks move along the rRNA genes (rDNA) at a uniform rate and terminate at multiple, apparently random sites [6, 71]. In contrast, a polar RFB is found at the 3’ end of the intensely transcribed rRNA genes of Xenopus cultured cells [72]. This suggested that the RFB is developmentally regulated, in parallel with rRNA transcription. In fact, up to 15 different RFPs simultaneously appear in the Xenopus rDNA at the midgastrula stage, when rRNA transcription abruptly increases [73]. They are found in both the TU and the IGS. They disappear during the neurula stage, except for a polar RFP at the 3’ end of the TU (at the same location as the RFB in cultured cells), which persists to the tadpole stage. Given that TTF-I has been involved in both replication arrest and chromatin remodeling in mouse rDNA, the transient appearance of multiple RFPs at midgastrula in Xenopus may reflect some chromatin remodeling process associated with the developmental activation of rRNA transcription. A regulated RFB has also been found in the rDNA of the ciliated protozoan Tetrahymena thermophila [74]. In this species, the rDNA is in the form of linear palindromes that are amplified during macronuclear differentiation. During vegetative replication, initiation occurs at one of the duplicated origins within the central IGS. In nondividing amplifying cells, multiple rounds of initiation occur within single rDNA molecules on both sides of the palindrome, and forks stall at a RFB located in the center of the palindromic rDNA. This RFB is absent in cycling vegetative cells, and the significance of this regulated RFB remains to be determined.

Mechanisms and consequences of replication fork arrest

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6. Replication arrest due to a stable RNA-DNA hybrid

8. Topology of arrested replication intermediates

It has long been known that it is difficult to generate palindromes involving ColE1 origins. The replication behavior of plasmids containing two unidirectional ColE1 replication origins in either the same or opposite orientations has been investigated using 2D gel electrophoresis [75, 76]. Replication initiates at only one of the two potential origins regardless of their orientation, a phenomenon known as origin interference. When the two ColE1 origins are inversely oriented, the accumulation of a specific replication intermediate is observed. The replication fork initiated at the active origin stalls at the inversely oriented silent origin. The inter-origin distance affects neither their ability to interfere with each other, nor to act as a RFP. Formation of a stable RNA-DNA hybrid at the origin is part of the normal initiation mechanism at the ColE1 origin and provides a 3’OH end that serves as a primer for DNA synthesis. These steps appear to take place at silent and at active origins, while the subsequent primer elongation and primosome assembly may or may not follow, for unknown reasons. The only element found to be essential for the silent origin to act as a RFP is the presence of a transcription promoter upstream of the origin. This suggests that a replication fork may pause when it encounters the 3’RNA end of a stable RNA-DNA hybrid at the silent origin. In vitro helicase assays indeed show that the E. coli DnaB helicase is unable to unwind RNA-DNA hybrids [76]. As DnaB moves in the 5’ to 3’ direction along the lagging strand template, it would find no obstacle when it approaches the silent origin from the other side, explaining why pausing only occurs if the two origins are inversely oriented.

8.1. Unlinking of DNA during replication As DNA is replicated, the unwinding of the parental strands by helicases causes compensatory overwinding, or (+)∆Lk, which must be removed by topoisomerases (reviewed in [78]). In principle, the (+)∆Lk could distribute over the entire partially replicated molecule, taking the form of positive supercoils in the unreplicated region and of positive intertwinings of the two replicated DNA segments (precatenanes). Initial electron microscopy (EM) observation of purified RIs from eukaryotes and prokaryotes showed supercoiling of the unreplicated DNA but no precatenane in the replicated region (the ‘butterfly’ conformation). Based on this observation, the prevailing view has been that topoisomerases only act on positive supercoils ahead of the forks. However, this cannot explain unlinking when only a short stretch of unreplicated DNA remains between converging forks. Studies of plasmid replication in E. coli instead suggest the coexistence of two modes of strand unlinking, with DNA gyrase actively introducing negative supercoils in the unreplicated region, and topoisomerase IV removing precatenanes in the replicated region and decatenating complete replication products [79, 80]. In the case of eukaryotic DNA replication, it is well known that either topo I or topo II can suffice for replication fork elongation. While topoisomerase I can relax positive supercoils ahead of the fork, the site of topoisomerase II action has not been clearly defined. Recent evidence suggests that topoisomerase II removes precatenanes but does not act in the unreplicated region during replication of plasmid DNA in a physiological chromatin context in Xenopus egg extracts (Lucas and Hyrien, unpublished).

7. Replication arrest due to transcription RFP sites have been mapped to the tRNA genes of S. cerevisiae in vivo [77]. These RFPs are polar, stalling replication forks only when they oppose to the direction of tRNA transcription. Mutant tRNA genes defective in assembly of transcription initiation complexes and a temperature sensitive RNA polymerase II mutant defective in initiation of transcription do not stall replication forks, suggesting that transcription is required for RFP activity. This distinguishes the tRNA-associated RFPs from the rDNA RFB, which is independent of transcription. The polar arrest at the tRNA-associated RFP could result from a direct interaction between the transcription and replication apparatus, from an interaction between the nascent tRNA and the replication fork, or from the accumulation of positive supercoils in the template between approaching replication and transcription complexes. The observation that the RFP zone is larger than the tRNA gene itself is more consistent with the latter model.

8.2. Topology of purified stalled RIs The structure of purified RIs was recently reexamined by electrophoresis and electron microscopy, using RIs accumulated in vivo or in vitro by Tus-induced arrest of replication of plasmids containing two Ter sites [81]. Topoisomers of stalled RIs have electrophoretic mobilities indicative of a precatenane conformation, and direct EM observation by a novel spreading technique shows DNA crossings in both the replicated and unreplicated regions. It seems that the previously used Kleinschmidt procedure artefactually alters the conformation of RIs such that they adopt a butterfly conformation. Thus, the ∆Lk is not confined to the unreplicated region but distributed throughout the molecule on purified RIs. It has not been formally demonstrated that a similar distribution of the ∆Lk occurs in vivo, but this would be consistent with the observed division of labor between gyrase and Topo IV during replication and with their substrate preferences [79, 80].

12 The stalled RIs have a (–)∆Lk. This is expected since they are produced in the presence of DNA gyrase. Transient RIs have not been examined so far, and it is unclear whether the activity of gyrase and topo IV is so potent that DNA remains negatively supercoiled during replication despite the production of (+)∆Lk by fork movement. In fact, net positive supercoiling may drive the fork backward and promote the reannealing of the nascent strands (Schvartzman, personal communication) [82], with potential implications for recombination at replication forks (see below). It is unknown whether RFBs, in addition to blocking the progression of replication forks, may act as topological barriers. If this were the case, a transient accumulation of positive supercoils may occur when a replication fork approaches a RFB. 8.3. Topology of stalled RIs probed in vivo by DNA knotting As mentioned previously, plasmids carrying two ColE1 replication origins in inverse orientation accumulate RIs in which the bubble spans the distance between the two origins. Strikingly, these accumulated RIs exist as a series of stereoisomers having one or more knots within the replicated portion [75, 76]. Electron microscopy of RecAcoated accumulated RIs shows that the knots formed contain predominantly positive signs of crossing [83]. This type of knots can be simply explained by the topoisomerase-mediated interlinking of overlapping coils of a replication bubble wound in a left-handed way. Since knotting events probe the actual topology and the resulting structure of DNA in vivo, these observations confirm that stalled RIs carry a (–)∆Lk in bacterial cells, and strongly suggest that a fraction of this (–)∆Lk takes the form of (–)precatenanes. It is unclear if the knotting of replication bubbles is associated with the arrest of replication forks. Transient RIs might have a different topology and be less prone to knotting or even carry knots with predominantly negative signs of crossing. 9. Replication arrest, recombination, and aging 9.1. Hyper-recombination at bacterial termini As previously mentioned, the function of replication arrest at bacterial termini is not obvious since tus can be deleted without affecting cell growth. Several observations associate the E. coli terminus with DNA recombination. Certain classes of RecA-dependent homologous recombination are 10–100 times more frequent in the terminus region than at other sites on the chromosome [84]. Some of these events are dependent on the Ter-Tus system. Specific sequences recognized by the RecBCD recombination enzymes are needed near the

Hyrien termini for the elevated rates of recombination. These data suggested that replication forks stalled at Ter sites may be efficient loading sites for the homologous recombination machinery. 9.2. Replication arrest and recombination in yeast rDNA Like the bacterial termination systems, the role of the rDNA RFB is not clear. As observed for the tus gene in E. coli, deletion of the FOB1 gene in S. cerevisiae abolishes the RFB activity but does not result in any obvious growth defects even after 116 generations [85]. Recently, Fob1 was implicated in the expansion and contraction of yeast rDNA repeats [86]. It has been observed that the absence of an essential subunit of RNA polymerase I in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number from ≈ 150 to ≈ 80, at a rate of approximately one copy per generation. Reintroduction of the missing RPA135 gene induces a gradual increase in repeat number back to normal level, at a similar rate. FOB1 is essential for both the decrease an the increase in rDNA repeats. It was suggested that replication arrest at the RFB stimulates DSB formation at the fork, and that repair between non-aligned sister chromatids results in expansion or contraction of the rDNA repeats. The equilibrium between deletion and expansion events results in a heterogeneous copy number, which is smaller in the absence of Pol I. The role of Pol I in shifting the equilibrium towards a higher repeat number is unclear. In contrast, fob1 mutants show a remarkable homogeneity in repeat number. Recently, deletion of the FOB1 gene has been demonstrated to extend the life span of yeast mother cells [85]. One cause of aging in yeast is the accumulation of extrachromosomal rDNA circles (ERCs) arising from the tandemly repeated chromosomal copies [87]. The accumulated ERCs may interfere with cell growth by titrating away some critical component of the replication or transcription machinery. The deletion of the FOB1 gene slows down the generation of these circles and thus extends life span. It was suggested that ERCs could arise by DSB at a stalled fork followed by intrachromatid recombination. These results suggest that recombination in the rDNA depends on replication. This is consistent with the discovery that Holliday junction recombination intermediates are only detected in S phase in the rDNA of wild-type cells, and that the level of these junctions is elevated in some replication mutants [88]. 9.3. Pathways linking replication arrest and recombination in E. coli A mechanistic link between replication arrest and homologous recombination has been established in E. coli by analysis of dnaB and rep mutants [89]. DnaB is the only essential helicase in E. coli. The Rep helicase is not

Mechanisms and consequences of replication fork arrest essential, but chromosome replication is slower in rep mutants. It has been observed that rep recB and rep recC mutants are non-viable at restrictive temperature [90]. This lethality, as shown using pulse-field gel electrophoresis, results from the accumulation of broken chromosomes [91]. Blockage of replication forks by a dnaBTS mutation, or insertion of an improperly oriented Ter site in the chromosome, also results in increased breakage in a strain carrying a recB mutation. However, little or no double-strand breaks (DSBs) are detected in single rep mutants, suggesting that the RecBCD enzyme either repairs or prevents DSBs following inactivation of the Rep helicase. The RecA protein and RecBCD complex are both essential for recombinational repair of DSBs, regardless of their origin. In vitro, RecBCD binds to DNA doublestranded ends, then unwinds DNA while simultaneously degrading it. Upon encountering a chi site, the polarity of degradation is switched from 3’-5’ to 5’-3’. This leads to the production of a 3’ single-stranded DNA, which is bound by RecA and invades a homologous molecule to initiate a strand exchange event [92, 93]. The RuvABC complex is implicated in the mechanism of chromosome breakage at arrested replication forks [89]. Mutations that inactivate the ruvAB operon suppress the non-viability of rep recBTS recCTS strains and the occurrence of DSBs at restrictive temperature. DSB formation in a dnaBTS recB strain and half of the spontaneous DSBs occurring in a recB single mutant are also RuvABC-dependent. Thus, paused replication forks are acted upon by RuvABC. RuvA and RuvB proteins are known to act in concert with RuvC in the late steps of homologous recombination (reviewed in [94]). RuvA binds specifically to Holliday junctions. RuvB is an ATP-dependent helicase which catalyses branchmigration of Holliday junctions in the presence of RuvA. RuvC is an endonuclease specific for Holliday junctions, which introduces symmetrical strand cleavage across the point of strand exchange. The double stranded end recognized by RecBCD in rep strains is created by the RuvAB complex, which is devoid of endonuclease activity, and not by the RuvC endonuclease. While inactivation of ruvAB rescues the viability of rep recBTS recCTS strain at non-permissive temperature, inactivation of RuvC renders this strain non-viable at any temperature. The requirement for RecBCD in rep ruvC strain indicates that the RecBCD target, a doublestranded end, exists in the absence of RuvC. In contrast, the rep ruvABC strain is viable in the absence of RecBCD, which indicates that RuvAB is necessary for double stranded end formation. The following model was suggested to account for these observations (figure 2) [89]. After arrest of a replication fork and disassembly of the replisome, a Holliday junction may form spontaneously by annealing of the two nascent strands. This step may be promoted by supercoil-

13 ing constraints, as explained in the previous section. RuvAB may bind to and promote branch-migration of the junction, forming a double stranded tail out of the replication fork. Normally, the RuvAB complex would be uncoupled from RuvC and the double stranded tail would be recognized by RecBCD. In a recA+ strain, encounter of a chi site would initiate a homologous recombination event, restoring a replication fork with limited DNA degradation. In a recA strain, the exoV activity of RecBCD would degrade the duplex DNA and remove RuvAB. Prior resolution of the Holliday junction by RuvC would release a chromosome arm that requires RecBCD and RecA for repair. In the absence of repair, breakage at the other fork may result in the observed linearization of chromosomes. It is important to stress some features of this model which are different from those proposed earlier to explain recombination events at the yeast rDNA locus. In this model, a Holliday junction and a double stranded end are formed at the stalled replication fork without actual DNA breakage. A DSB only ensues if RuvC acts prior to RecBCD. Thus, the normal role of the recombination machinery at the fork might be to prevent DNA breakage rather than to repair it. This model could explain why the accumulation of Holliday junctions in the rDNA of yeast replication mutants occurs via a RecA-homologue independent mechanism [88]. It may also explain why the frequency of mitotic recombination and extrachromosomal circle formation in the yeast rDNA is strongly increased in mutants with low topoisomerase activity [95, 96], since a deficient removal of (+)∆Lk generated during replication or downstream of the rRNA transcription machinery might facilitate the formation of Holliday junctions at the forks. Finally, replication blockage has also been associated with illegitimate recombination. The analysis of deletions occurring in E. coli on a plasmid bearing two Ter sites in opposite orientations suggests the existence of two distinct mechanisms [97]. The first recombination process joins sequences preceding the two Ter sites and showing no homology, and depends on topoisomerase I activity. The second process joins sequences of 3–10 bp homology, and depends on exoV activity but not topoisomerase I activity. It is tempting to speculate that other kinds of replication roadblocks may trigger illegitimate recombination. In higher eukaryotes, illegitimate recombination appears to be more active than homologous recombination, and some genome rearrangements such as gene amplification events have been proposed to occur by recombination at replication forks, with the possible implication of topoisomerase 1 [98, 99]. Thus, replication arrest may be a key factor in genome instability. Further work is required to learn to which extent the mechanistic insights gained from studies with microorganisms have their counterparts in higher eukaryotes.

14

Hyrien

Figure 2. Model for recombination events at replication forks (adapted from [89]). A. A stalled replication fork. B. Formation of a Holliday junction by spontaneous reannealing of the parental strands and de novo annealing of the nascent strands. C. Binding and further branch-migration of the Holliday junction by RuvAB. D. Binding and processing of the double strand end by RecBCD. E. RecA-mediated strand-exchange intermediate. F. Resolution by RuvC and rescue of the replication fork. G. In the absence of RecBCD action, resolution of the junction by RuvC releases a chromosome arm that requires RecBCD and RecA for repair. H. In the absence of RecA action, RecBCD degrades the double stranded end up to the junction. Replication can restart when RecBCD removes RuvAB.

Mechanisms and consequences of replication fork arrest Acknowledgments I thank K. Marheineke and I. Lucas for reading the manuscript. This work was supported in part by the Association pour la Recherche sur le Cancer.

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