Current Biology 24, R445–R452, May 19, 2014 ª2014 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2014.03.060

Polymerase Stalling during Replication, Transcription and Translation Ellen R. Edenberg, Michael Downey, and David Toczyski*

Replication, transcription, and translation stress all lead to stalling of their respective polymerases (DNA polymerase, RNA polymerase, and the ribosome), and the cell must respond to these events in order to preserve macromolecular integrity. In response to replication stress such as DNA damage, the cell activates a checkpoint and promotes repair or bypass at the lesion. Transcriptional stress leading to stalling of RNA polymerase can also be caused by DNA damage, and recognizing stalled RNA polymerase can lead to transcription-coupled repair or, in response to prolonged stalling, degradation of the polymerase. Translational stress generated by problems either with the mRNA template or the ribosome itself also leads to stalling of the ribosome, and the cell responds by degrading both the message and the nascent polypeptide. In this review, we will discuss the stresses that lead to stalling of each of the polymerases and how the cell recognizes and responds to the stalled enzymes. Introduction The central dogma of molecular biology describes the transfer of information from DNA to RNA to protein. The synthesis of each of these molecules is carried out by a large multisubunit polymerase: DNA polymerase, RNA polymerase, and the ribosome (in this review, we will include the ribosome when referring to polymerases in general). In each case, issues can arise causing these polymerases to inappropriately stall on their respective templates, and the cell must identify and correct such events to preserve genomic and macromolecular integrity. Polymerases can stall for several reasons. First, the polymerase may attempt to read a damaged template, blocking its progression. By recognizing the damaged template, and promoting the repair or destruction of that template, each of the polymerases has a template scanning, protective function. Alternatively, the polymerase itself can be damaged or incorrectly assembled, causing it to stall or create an aberrant product. In addition, barriers on templates, such as protein complexes or secondary structures, can also impede the progress of polymerases. Finally, low nucleotide or amino acid pools cause polymerase stalling. In each of these cases, cells must distinguish between an actively replicating polymerase and a stalled polymerase trying to read or repair a damaged template. One of the least understood aspects of the response to polymerase stalling is the actual recognition of this event. The nature of DNA synthesis, RNA synthesis and translation places unique restrictions on the ways in which the cell can correct problems occurring during each phase of macromolecular synthesis. In the case of DNA or RNA polymerase, the template, DNA, must be repaired. For DNA polymerase, not only is its substrate essential, but the nascent

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. *E-mail: [email protected]

Review

polymerase product must also be maintained in a manner that allows replication to be restarted after repair. In contrast, the product of any single RNA polymerase event is nonessential. The stalled RNA polymerase itself can be targeted for destruction, and the DNA template can return to the ground state after the nascent message and polymerase are removed. During translation, the ribosome, nascent polypeptide and mRNA are all, in theory, dispensable, although an effort to conserve resources may restrain the degree to which these components are degraded. In this review, we will examine the common elements of pathways that react to each of these situations, as well as contrasts between them. DNA Replication Replication is one of the primary ways that a cell scans its genome for damage. During replication, DNA polymerase associates with a large number of auxiliary factors, including the MCM replicative helicase complex and the sliding clamp PCNA, to processively move along the DNA. In addition to its polymerizing activity, this complex coordinates the damage response by activating the DNA replication checkpoint and promoting repair. Consistent with a template scanning role for DNA replication, non-cycling cells do not repair genome-wide DNA damage as well as cells that are actively cycling [1]. Moreover, damaging agents such as methyl methanesulfonate (MMS) generate modified bases that are largely detected when the polymerase encounters the lesion [2]. The replication checkpoint is activated at replication forks stalled by DNA damage, depletion of nucleotide pools (for instance with ribonucleotide reductase inhibitor hydroxyurea (HU)), or inhibition of polymerase itself (with drugs such as aphidicolin). In addition, replication through some sequences is inherently problematic for the polymerase. Loci called common fragile sites (in mammalian cells) or replication slow zones (in budding yeast) are sensitive to replication stress and prone to breakage. The stability of these sites in yeast and mammals requires the DNA replication checkpoint kinase Mec1/ATR (for this review, homologs will be referred to by budding yeast/mammalian names), suggesting that these sites elicit damage after polymerase stalling during S phase that is recognized by the checkpoint [3,4]. DNA polymerase can also stall at specific protein barriers or at highly transcribed genes [5]. Replication fork barriers (RFBs) in Schizosaccharomyces pombe and Saccharomyces cerevisiae are composed of DNA binding proteins that cause unidirectional polymerase stalling [6,7]. The block at RFBs requires the budding yeast replication checkpoint proteins Tof1 and Csm3 (Swi1 and Swi3, respectively, in S. pombe) which associate with MCMs and move with the fork, suggesting that stably stopping at RFBs is an active process [8–10]. Stalling at such physical barriers may differ from stalling at DNA lesions or due to nucleotide depletion because, in addition to blocking the polymerase, the MCM helicase is also directly blocked. Consistent with this notion, stalling at RFBs does not lead to the accumulation of single-stranded DNA (ssDNA) [11] and does not require the DNA replication checkpoint pathway [9,12].

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Activation of the DNA replication checkpoint affects many aspects of cellular metabolism, including halting the cell cycle and promoting survival [13]. Upon recognition of DNA damage or polymerase stalling, the replication checkpoint sensor kinase Mec1/ATR is recruited to sites of stalled polymerase and activated. This kinase then phosphorylates and activates downstream effector kinases, importantly Rad53 (in budding yeast) and Chk1 (in mammals). While forms of DNA damage that generate large regions of ssDNA are recognized throughout the cell cycle (such as UV damage which is processed by nucleotide excision repair machinery [14]), genotoxic agents that cause damage or stall polymerase and are recognized during S phase rely on the replisome component Mrc1/Claspin as the adaptor for checkpoint activation. Mrc1/Claspin associates with Tof1/Timeless and Csm3/Tipin and directly binds the downstream checkpoint kinases and promotes their activation. The signal for activation of the DNA replication checkpoint is the accumulation of exposed ssDNA coated with the ssDNA binding protein RPA. In budding yeast, approximately 200 nucleotides of ssDNA are exposed during normal replication, whereas forks stalled by HU generate about 320 nucleotides [15]. The additional exposed ssDNA at these structures distinguishes a stalled fork from ongoing replication. Importantly, ongoing replication itself does not activate the canonical checkpoint, and budding yeast cells can enter anaphase in the presence of actively replicating DNA [16]. RPA-coated ssDNA activates the checkpoint by providing a template for the co-recruitment of the upstream checkpoint kinase Mec1/ATR and its activators [17,18]. Mec1/ ATR is recruited to RPA by its binding partner Ddc2/ATRIP. The downstream kinase (Rad53 or Chk1) is recruited through interactions with the mediator Mrc1/Claspin. Highlighting the importance of increased local protein concentrations achieved via these recruitment events, forced co-localization of Mec1/ATR with its activators or with Mrc1/Claspin is sufficient for in vivo checkpoint activation even in the absence of DNA damage [19,20]. Once the checkpoint is activated, it maintains the replication competence of stalled forks and prevents the firing of later origins (Figure 1). Firing of these origins is inhibited by the checkpoint in both yeast and mammalian cells. In budding yeast, Rad53 phosphorylates and inhibits replication initiation factors Dbf4 and Sld3 [21,22], and both the inhibition of Treslin (the ortholog of Sld3) and the phosphorylation of Dbf4 by checkpoint kinases is conserved in mammals [23,24]. The MCM replicative helicase complex and associated proteins are also phosphorylated by checkpoint kinases [25,26]. Although the effects of these particular phosphorylations are not well understood, Chk2 phosphorylation of the MCM helicase complex in flies blocks its activity [27]. The checkpoint’s most important function has canonically been thought to be the stabilization of the replication fork itself — preventing DNA breaks from accumulating by promoting the continued association of polymerase and other replication factors (called the replisome) with each other and with the DNA. Recent work, however, suggests that the replisome is stably maintained at the replication fork even in the absence of a functional checkpoint [25]. This study suggests that the checkpoint may primarily function to maintain the replisome in a replication-competent state at stalled forks, in order to allow replication to resume once the challenge has been removed. In yeast, deletion of the

nuclease Exo1 can suppress the sensitivity of rad53D mutants to MMS, suggesting that inhibiting Exo1-dependent processing may be one important checkpoint function [28,29] (Figure 1). In mammals, HARP/SMARCAL1 is recruited to sites of DNA damage by a direct interaction with RPA [30–33], and phosphorylation by the checkpoint kinase ATR limits its activity [34]. SMARCAL1 is a DNA annealing helicase that can re-wind ssDNA stably coated with RPA, as well as perform branch migration and fork regression [35,36], which suggests that it may be a checkpoint target involved in both fork stabilization and replication restart [30,31,34,37]. An intriguing new study in mammalian cells suggests that DNA breaks accumulate at a fork following exposure to replication stress because an excess of ssDNA exhausts the pool of RPA [38]. The data show that forks are initially stable, even in the absence of a functional checkpoint, and are prone to breakage only after RPA pools are exhausted. Moreover, modulating levels of functional RPA can speed up or slow down this process. This paper suggests that the checkpoint inhibition of late origin firing and potentially the inhibition of the MCM helicase or other targets involved in replication that limit ssDNA accumulation may indirectly allow the checkpoint to maintain stable forks for prolonged periods of time. The stalled replisome can also directly coordinate repair through the modification of the sliding clamp PCNA (Figure 1). In response to DNA damage, PCNA is modified by ubiquitination. Ubiquitination of PCNA is independent of the checkpoint, but it is promoted in response to polymerase stalling by the same upstream signal — accumulation of RPA-coated ssDNA [39,40]. In this case, Rad6 and Rad18 are recruited to RPA and monoubiquitinate PCNA at lysine K164 [41]. Monoubiquitination of PCNA promotes bypass of damage by the translesion polymerases Polh, Polz, and Rev1 [42,43]. These polymerases have larger active sites and no proofreading, so they can accommodate bulky adducts and polymerize across them. Although often thought of as error-prone, they bypass some DNA lesions in an error-free way, for instance by adding adenosines across from thymine dimers. In yeast, monoubiquitination of PCNA at K164 can be extended by the Ubc13–Mms2 heterodimeric E2 and the Rad5 E3 to generate K63-linked polyubiquitin chains [41]. Polyubiquitinated PCNA has been proposed to promote error-free lesion bypass through a template-switching mechanism. Recent work in mammalian cells has demonstrated that polyubiquitinated PCNA promotes fork restart through the recruitment of ZRANB3. ZRANB3 has several biochemical activities — as an annealing helicase, a translocase, and a structure-specific endonuclease — that might allow error-free template bypass, fork restart, or even, in the case of its endonuclease function, repair of DNA damage at the fork [44–46]. Mammals have two Rad5-related enzymes (HLTF and SHPRH). Overexpression of these enzymes has been shown to promote polyubiquitination of PCNA [47,48]. However, these enzymes also coordinate translesion synthesis, and HLTF promotes monoubiquitination of PCNA [49]. The precise mechanism of polymerase switching from the processive replicative polymerases to translesion polymerases is still unknown, though translesion synthesis can occur without bulk DNA replication [50]. A recent paper suggests a model for the polymerase switch by showing that the catalytic subunit of the lagging strand polymerase (Pol3, part of polymerase d)

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Template switching

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Figure 1. Polymerases coordinate the response to stalling. When DNA polymerase (top, depicted in blue) stalls, ATR is recruited to RPA-coated singlestranded DNA (ssDNA). ATR then phosphorylates and activates Rad53 (yeast) or Chk1 (humans), which goes on to phosphorylate downstream targets including Dbf4 and Sld3/Treslin to inhibit new origin firing, and, potentially, Exo1. RPA-coated ssDNA can also recruit Rad18–Rad6 to monoubiquitinate PCNA, which promotes association of translesion polymerases (TLS Pol, depicted in teal). Rad5-dependent polyubiquitination of PCNA promotes error-free lesion bypass by a template-switching mechanism. RNA polymerase (middle, depicted in fuchsia) stalling can promote transcription-coupled repair by backtracking and recruiting Rad26/CSB and TFIIH. Alternatively, after prolonged stalling, RNA polymerase can be degraded as a mechanism of last resort. Def1 is processed in the cytoplasm following monoubiquitination by Rsp5, which allows Def1 to accumulate in the nucleus. Def1 then recruits an Elongin– Cullin E3 ligase to polyubiquitinate RNA polymerase II to promote its degradation. Stalling of ribosomes (bottom, shown in red) leads to Hbs1 and Dom34/Pelota promoting dissociation of the ribosome and an endonuclealytic cleavage of the mRNA, which is then fully degraded by exonucleases Xrn1 and the exosome. Ltn1 then associates with the large subunit and ubiquitinates the nascent polypeptide to target it for degradation. Polyubiquitin chains depicted in green reflect degradative chains, while polyubiquitin chains depicted in purple are non-degradative, K63-linked chains.

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is degraded in response to DNA damage [51], which may allow translesion polymerase Rev1 to interact with the non-catalytic subunits of polymerase d. Interestingly, the process of polymerase degradation requires Def1, which associates with Rad5 [51] and is known to promote the degradation of RNA polymerase (discussed below). These data seem to contradict other work suggesting that the replisome is stable following replication stress [25]. In trying to reconcile these recent papers, we note that replisome stability was shown with leading strand polymerase (Pol ε) rather than Pol d. The two papers also use different agents to stall replication, and the degradation of Pol3 was never shown to occur at stalled replication forks themselves. Transcription RNA polymerase responds to the same lesions on the same template as DNA polymerase. In fact, stalling of transcription by various means can result in RPA- and ATR-dependent phosphorylation of p53, suggesting activation of the checkpoint [52]. An elongating RNA polymerase will not stall at all DNA lesions, but is particularly susceptible to pausing at bulky DNA adducts, such as cisplatin-induced crosslinks, and UV-induced photoproducts, such as thymine dimers [53]. Detailed structural work has provided

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clues as to how RNA polymerase stalls at different DNA lesions. Cisplatin-induced DNA damage generates a conformational change in the template strand that prevents the lesion from entering the RNA polymerase II active site [54]. In contrast, a thymine dimer can enter the active site, and subsequent stalling depends on which ribonucleotide is inserted to base-pair with the lesion [55]. Similar to translesion DNA polymerases, transcription machinery preferentially inserts adenylate across from these sites, which is error-free and does not result in stalling. Instead, stalling in this situation only occurs when a uridylate nucleotide is mis-incorporated [55,56]. More work is needed to know whether these examples represent two common modes of polymerase stalling or whether other lesions stall RNA polymerase by distinct mechanisms. Also, it will be important to determine whether the mechanisms of RNA polymerase stalling described in these studies, which have been done on naked DNA, are affected by DNA binding proteins such as nucleosomes or RPA. Lesions that are bypassed

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by RNA polymerase can be subsequently repaired by canonical repair machinery or recognized by the stalling of DNA polymerase during replication. While the mechanisms promoting trans-lesion RNA synthesis at thymine dimers and other lesions remain poorly understood, it is clear that bypass can be important for cell survival after DNA damage, albeit increasing transcription-associated mutagenesis under some circumstances [56]. Similar to DNA polymerase, RNA polymerase stalling can lead to lesion processing and DNA repair [57] in a process called transcription-coupled repair (TCR). TCR was first described almost three decades ago [58,59] and is largely studied in the context of thymine dimers and other lesions caused by UV, as these lesions can stall RNA polymerase for prolonged periods of time. One result of TCR is that lesions in the template DNA strand are repaired with increased efficiency relative to those occurring in the nontranscribed strand [60]. A key question in TCR is how repair machinery gains access to the DNA lesion, which may be obstructed by RNA polymerase enzyme. In eukaryotic cells, only RNA polymerase I has been observed to be released from sites of DNA damage encountered during transcription [61], and this phenomenon is poorly understood. Instead, eukaryotic RNA polymerase II backtracks along DNA to facilitate endonuclease access [62]. A recent study [63] suggests that backtracking may also play a role in providing access to repair machinery in bacteria, although prokaryotic RNA polymerase can also be released altogether [64]. Backtracked eukaryotic RNA polymerase itself cleaves the transcript to allow its realignment with the polymerase active site. This cleavage activity is stimulated by TFIIS and is required for viability in yeast [65]. In many ways, TCR is very similar to nucleotide excision repair (NER), which recognizes the same lesions through direct recognition of structural aberrations in the DNA template. In both pathways, recruitment of TFIIH to damage sites provides a scaffold that directs the XRCCI and XPA nucleases to excise the lesion along with short stretches of surrounding sequence (Figure 1). During TCR, the first responder, Rad26/CSB, recruits TFIIH. How Rad26/CSB recognizes a stalled RNA polymerase to signal the presence of a DNA lesion is unclear [62]. Some evidence suggests that Rad26/CSB may transiently associate with RNA polymerase II during transcriptional elongation and that prolonged stalling triggers a more stable interaction [66]. In mammalian cells, the CSA protein, which has E3 ubiquitin ligase activity, is also required for scaffold assembly. A ubiquitin binding domain on CSB is required for TCR in mammalian cells, despite the proficient recruitment of lesion processing machineries to sites of stalled RNA polymerase II in its absence [67]. These data suggest that recruitment of repair factors to DNA lesions may not be enough to trigger DNA repair and that a second, as yet unknown, signal is required to initiate the process. Whether this unknown signal also recognizes stalled polymerase is still unknown. Mutations in CSA and CSB give rise to Cockayne syndrome, a disease characterized by sensitivity to UV light, progressive defects in neurological development and premature aging. These phenotypes differ from those associated with global defects in NER, and this, and other recent work, suggests that Cockayne-related phenotypes stem from deficiencies in transcribing specific genes rather than from a global defect in DNA repair (reviewed in [62,68]).

RNA polymerase II itself can be degraded by the ubiquitin– proteasome system as a mechanism of last resort in response to prolonged stalling [69] (Figure 1). Ubiquitination of the largest RNA polymerase II subunit, Rpb1, is a two-step process [70]. Monoubiquitination, catalyzed by the Rsp5 E3 ubiquitin ligase (NEDD4 in humans) [71], occurs at significant levels even in the absence of DNA damage or transcriptional stress [69,72]. Prolonged transcriptional pausing leads to the extension of this monoubiquitin mark to K48-linked polyubiquitin chains that signal RNA polymerase II for degradation. The polyubiquitination of RNA polymerase II occurs via an Elongin–Cullin E3 ligase containing Elc1, Ela1, and Cul3, which is recruited to the stalled polymerase by the Rpb1binding protein Def1. Elegant work from the Svejstrup lab has shown that an amino-terminal CUE domain in the Def1 protein binds to a ubiquitin homology domain on Ela1 to facilitate association of the Elongin–Cullin complex with Rpb1 [73]. How does Def1 specifically recognize stalled RNA polymerase II rather than molecules that have initiated transcription but remain poised for elongation downstream of transcription start sites? Selection of Rpb1 as a substrate depends on its carboxy-terminal domain (CTD). The RNA polymerase II CTD includes 26 repeats of a heptapeptide sequence in yeast and 52 such repeats in humans. The phosphorylation pattern of the CTD provides a molecular signature for polymerase molecules during the initiation and elongation phases of transcription. In yeast, RNA polymerase II ubiquitination has been shown to be negatively regulated by CTD phosphorylated on serine 5 of its heptapeptide repeats [74], which is characteristic of promterassociated polymerases [75]. In contrast, the human Elongin complex has been suggested to selectively target RNA polymerase II with serine 5 phosphorylated [76]. As suggested previously, perhaps other post-translational modifications on the CTD distinguish between elongating polymerases that have stalled and initiated polymerases intentionally poised downstream of the transcription start site [69]. It is noteworthy, for example, that the terminal CTD repeats on human Rpb1 have been found to be acetylated [77]. Confining Rpb1 ubiquitination to obstructed polymerases, rather than elongating polymerases, seems to be regulated by Def1 itself. Def1 cycles between the nucleus and the cytoplasm, and its nuclear retention is promoted by a proteasome-mediated cleavage event that removes a carboxyterminal nuclear export signal [73]. Treatment with agents causing DNA damage or inhibitors of transcription elongation leads to rapid Def1 processing, which is dependent on monoubiquitination of Def1 by Rsp5, the same ligase that targets Rpb1 [73]. The signals that direct this event remain unclear. The deubiquitinating enzyme Ubp2, which binds to Rsp5 in vivo and is involved in regulating Rpb1 ubiquitination [78], can inhibit the accumulation of monoubiquitinated Def1 in vitro, possibly through the direct inhibition of Rsp5 activity [79,80]. It is tempting to speculate that prolonged stalling of transcription machineries may somehow inhibit Ubp2 activity, and thus promote Rsp5 function. Def1 also interacts with Rad26 [81], suggesting that factors involved in TCR could promote Rpb1 degradation if repair cannot occur. Regardless, much like the events that function to recruit repair proteins following an interruption to DNA replication, ubiquitin signaling cascades play an essential and complex role in mediating the response to prolonged stalling of RNA polymerase.

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Translation Unlike replication and transcription, in the case of protein biosynthesis, the mRNA template, the protein product, and the polymerase (ribosome) can all be subject to degradation. Translation can be disrupted by many of the same things that disrupt DNA replication and transcription. As with low nucleotide pools during replication and transcription, low amino acid levels stall translation. This is best characterized in prokaryotes, where, during attenuation (in the case of specific operons) or the stringent response, ribosome stalling signals changes in gene expression [82]. Moreover, many agents that induce DNA damage can also alter RNA, and this directly affects translation. Human cells treated with damaging agents or transfected with in vitro oxidized mRNA generate carboxy-terminally truncated translation products [83]. These likely arise when ribosomes stall, and the translation products themselves are thought to be targeted for degradation by ubiquitination, as they accumulate to high levels when the proteasome is inhibited. While enzymes that repair RNA have been described, many damaged templates are likely destroyed. Problems in translation can also arise on templates as a result of the sequences of the mRNAs themselves. This can take the form of errors in splicing or polyadenylation, mRNA truncation, transcriptional errors, or aspects intrinsic to the sequence, such as polyA stretches or a propensity to form secondary structures. In each of these cases, both the mRNA template and the nascent protein product are targeted for degradation. Three pathways have been described to recognize these mRNAs and nascent proteins depending upon the nature of the template problem: nonsense-mediated decay (NMD), no go decay (NGD) and non-stop decay (NSD) [84]. NMD is the outlier of these in that translation, per se, is not inherently problematic. Instead, NMD targets situations in which the open reading frame encoded by the mRNA is prematurely ended. NMD is triggered when a termination event occurs upstream of the last exon junction (as recognized by the existence of the exon junction complex) and by other more poorly characterized features. While this recognition was first believed to take place in the initial pioneer round [85], more recent studies suggest that it may also occur during subsequent rounds of translation [86]. Either way, it appears that, similar to replication and transcription, translation provides a template-scanning role. While initially thought to be distinct, NGD and NSD appear to be highly related mechanistically. NSD occurs when no termination codon exists and translation extends into the poly A tail, which codes polylysine and leads to ribosome stalling [87,88]. NGD is triggered by the direct pausing of the ribosome [89]. This can be due to a strong secondary structure in the message, by a truncated mRNA, or by some sequence elements, such as moderately long stretches of basic residues within the open reading frame as would be encountered upon translation of the poly A tail. As a consequence of ribosome stalling, the mRNA is targeted for degradation. Dom34/Pelota and Hbs1, which facilitate the degradation of the mRNA, are related to the translation release factors eRF1 and eRF3, respectively, and, analogously, bind to the ribosomal A site [89–91]. Dom34/Pelota and Hbs1 promote an endonucleolytic cleavage of the mRNA, which is then fully degraded by two exonucleases: Xrn1 and a nuclease complex called the exosome [84] (Figure 1).

Meanwhile, the nascent polypeptide that is generated during NGD and NSD is also targeted for degradation by ubiquitination through the action of a ubiquitin ligase called Listerin (Ltn1) [92]. Ltn1, together with two other factors, Tae2 and Rqc1, forms a complex that recognizes stalled ribosomes during NSD and NGD [92–94]. Hbs1 promotes the dissociation of stalled 80S ribosomes into 60S and 40S subunits [95–97]. Because Ltn1 appears to bind specifically to the 60S ribosomal subunit associated with the nascent peptide (as opposed to the intact 80S ribosome), elimination of Hbs1 blocks the binding of Ltn1 and the subsequent ubiquitination of the nascent peptide [98]. The Ltn1 complex also interacts with Cdc48, an AAA ATPase which binds and extracts ubiquitinated substrates from macromolecular complexes, and is therefore likely involved in fully removing the ubiquitinated nascent peptide to enable its destruction by the proteasome [93,94]. Together, these recent findings allow us to start to form a model for the way in which stalled ribosomes are processed, but we are still left without an explanation as to how they are recognized in the first place. Most analyses of stalled ribosomes have examined stalling occurring due to issues with the template; however, defective ribosomes can also be a source of translational stress. While this could, in theory, be due to misfolding, loss or damage of ribosomal proteins, most of the experiments examining this phenomenon thus far have investigated the effects of mutated or incorrectly processed rRNA. rRNA quality control is monitored quite closely. Inactivating point mutations in 18S rRNA result in its instability. Both mutant 18S rRNA and NGD intermediates are localized to P bodies, and the instability of the mutant 18S rRNA requires active translation and genes that also function in NGD, such as DOM34 [99]. This suggests that ribosomes containing defective 18S rRNAs stall and are recognized by similar machineries that recognize functional ribosomes that are stalled on aberrant messages. The final steps in processing of the 18S rRNA of the 40S subunit are preceded by a faux translation cycle in which the incomplete 40S and 60S form a translation-incompetent 80S-like particle, which is subsequently dissociated, helping to ensure that the 40S particle is properly assembled [100]. Surprisingly, ribosomes with mutant 25S rRNA (the yeast 28S) appear to be recognized and targeted by a distinct, more poorly understood, pathway [101]. Conclusions A number of common themes arise during the cellular responses to stalling during replication, transcription and translation. First, stalling of all three polymerase enzymes can be triggered by structural aberrations of the nucleic acid template. In addition, depletion of building blocks (dNTPs, NTPs, or amino acids) can stall polymerases. Since both DNA and RNA polymerase read the same template, these two polymerases have the most similarities, with stalled RNA polymerase even being capable of activating a checkpoint response [52]. In addition, RNA polymerase backtracking at sites of damage may allow transcriptional restart, similar to replication fork reversal at sites of stalled DNA polymerases [102]. Ubiquitin signaling cascades play a particularly important role at stalled DNA and RNA polymerases. In both cases, these signaling cascades are activated directly at the sites of stalling and recruit repair machinery, and may therefore limit signaling for repair to the lesion itself. Beyond its role

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in signaling, ubiquitin can also target each of the three polymerases for destruction. Importantly, the recent discovery that Def1 may play a role in mediating DNA polymerase ubiquitination after replication stress, in addition to its established role in targeting stalled RNA polymerase for destruction, suggests that stress may activate a common set of mediators. Other signaling proteins may also be shared between stalled DNA polymerase and stalled RNA polymerase. Recently, PRP19, a factor known to normally interact with RNA polymerase during unperturbed transcription [103,104], was found to promote ATR signaling in mammalian cells in response to DNA damage [105]. Perhaps this protein is also required for the cellular response to stalled RNA polymerase. Finally, for RNA and DNA polymerases that stall at sites of DNA damage, it is noteworthy that repair of the lesion itself is not sufficient to allow resumption of transcription or replication. In the case of DNA replication, chromatin structure and nucleosome re-assembly may be involved, and K56 acetylation on histone H3 is required [106]. In addition, turning off the checkpoint is itself an active process. Because of the broad cellular consequences of checkpoint activation, including the canonical effects on cell cycle progression, recovery is likely an obligatorily global phenomenon, involving dephosphorylation of checkpoint targets by several phosphatases. Transcriptional restart also appears to be an active, albeit poorly characterized, process. Intriguing work from the Almouzni group suggests that in mammals, transcription restart may be primed by the deposition of an H3 histone variant [107]. In yeast, the FACT nucleosome remodeling complex may play an analogous role by regulating H2A/ H2B deposition near stalled RNA polymerases [108]. In the case of transcription, the influence of chromatin may be very local. Although dissociated ribosomes released from their problematic templates would be free to re-initiate translation at any message, ribosome restart on the same template after stalling has not been characterized. Future work will be required to have a better mechanistic understanding of these processes. Acknowledgements We would like to thank Theresa Berens, Jesper Svejstrup, Kevin Mark, Alain Jacquier, and Micheline Fromont-Racine for comments on the manuscript. This work was supported by the National Institutes of Health grant GM059691 (D.P.T.).

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