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trough containing chocolate (followedby the rat consuming the chocolate), orby the rat traveling down the arm toencounter chocolate (on its own) andconsuming it [1]. Thus, familiarity ofhaving received chocolate (semanticmemory) was identical in bothinstances. But the source (episodicmemory) of the chocolate (‘directed’placement by the experimenter, or‘discovered’ by the rat) was different.

In Experiment 1, the place wherethe rat discovered chocolate on itsownwas replenished with chocolate onthe test, but not the place where therat was placed in front of the troughcontaining chocolate. Rats accuratelyremembered this source rule. Inanother experiment (Experiment 4),the rule was reversed and the ratsaccurately remembered this oppositesource rule — showing that handling(the rats) was not an artifact thatproduced a negative result (disruptedmemory). In Experiment 2, rats wereshown to accurately transfer theappropriate source rule to a second,different maze in which they had noprevious ‘source’ training — therebydemonstrating that overlearned cuesfrom a particular maze were not anartifact that produced a positive result(good memory).

Additionally, the rats’ sourcememory was shown to be special(Experiment 3) — as it should be if itreally was episodic memory — bylasting much longer (seven days ormore) than ‘run of themill’ memory (oneday) for regular rat chow. And lastly, therats’ source memory was shown(Experiment 5) to be disrupted bytemporarily disabling (with lidocaine)

a brain area (CA3 region of thehippocampus) thought to be crucial foraccurate human source memory. Thislast result adds important convergingevidence that this procedure really istesting something very close to sourcememory.

This study [1] sets the stage forexploring and better understanding theneural basis of source memory (andepisodic memory), not possible withhumans even with high-resolutionimaging. In addition to the CA3 regionof the hippocampus investigated in thiswork, the role of other brain areas couldbe tested in future experiments withthis procedure. Studies have shown theimportance of other medial temporallobe structures in relatedmemory tasks(for example, the CA1 region of thehippocampus, dentate gyrus,parahippocampal and entorhinalcortecies) [6]. But such memories arenot ‘stored’ in the medial temporallobes. Memories are distributed (neuralcircuits). Often (maybe always)remembering reactivates sensoryassociation areas (for example, parietallobe, located dorsal to the temporallobe) and even primary sensory areas(for example, occipital lobe locatedcaudal to the parietal lobe) thatproduced those memories in the firstplace [7].

Often (maybe always) what controlsreactivation of a memory comes froma very different brain region — theprefrontal cortex of the frontal lobe [7].So, reactivation of memories coupledwith reactivation along paths of theoriginal activation form loops ofmemory activity. With a rat model ofsource memory like that shown by

Crystal et al. [1], neural firing in severalbrain regions could be recorded in realtime as the rat makes correct (andincorrect) source judgments. Thesetechniques along with others (e.g.,molecular, genetic) may somedaybe able to specify pathways andmechanisms of how episodic memoryworks, perhaps leading to approachesfor repairing memory when it beginsto fail.

References1. Crystal, J.D., Alford, W.T., Zhou, W., and

Hohmann, A.G. (2013). Source memory in the rat.Curr. Biol. 23, 387–391.

2. Gardiner, J.M. (2002). Episodic memory andautonoetic consciousness: a first-personapproach. In Episodic Memory, A. Baddeley,M. Conway, and J. Aggleton, eds. (Oxford:Oxford University Press), pp. 11–30.

3. Clayton, N.S., Griffiths, D.P., Emery, N.J., andDickenson, A. (2002). Elements of episodic-likememory in animals. In Episodic Memory,A. Baddeley, M. Conway, and J. Aggleton, eds.(Oxford: Oxford University Press), pp. 232–248.

4. Tulving, E. (2002). Episodic memory andcommon sense: how far apart? In EpisodicMemory, A. Baddeley, M. Conway, andJ. Aggleton, eds. (Oxford: Oxford UniversityPress), pp. 269–287.

5. Mitchell, K.J., and Johnson, M.K. (2009).Source monitoring 15 years later: What havewe learned from fMRI about the neuralmechanisms of source memory? Psychol. Bull.135, 638–677.

6. Ranganath, C. (2010). Binding items andcontexts: The cognitive neuroscience ofepisodic memory. Curr. Dir. Psychol. Sci. 19,131–137.

7. Rissman, J., and Wagner, A.D. (2012).Distributed representations in memory: insightsfrom functional brain imaging. Annu. Rev.Psychol. 63, 14.1–14.28.

Department of Neurobiology and Anatomy,University of Texas Medical School atHouston, 6431 Fannin Street, Houston,TX 77030, USA.E-mail: Anthony.A.Wright@uth.tmc.edu

http://dx.doi.org/10.1016/j.cub.2013.01.055

Chromosome Biology: ConflictManagement for Replication andTranscription

A recent study has uncovered a new mechanism that attenuates DNAreplication during periods of heightened gene expression to avoid collisionsbetween replication and transcription.

James M. Dewarand Johannes C. Walter

In all organisms, chromosomes hosttwo essential metabolic process, gene

transcription and DNA replication,whichwould appear to conflict with oneanother. DNA replication copies thegenetic information in preparation forcell division and is initiated at sites

called origins [1]. At each origin, tworeplisomes are established that consistof a replicative DNA helicase, leadingand lagging strand DNA polymerases,and many accessory factors. The tworeplisomes travel away from the originin opposite directions, copying bothstrands of the duplex as they go. Whileprokaryotes generally replicate theirgenomes from a single origin,eukaryotic cells employ up to hundredsof thousands of origins in everyS phase. Like DNA replication,transcription involves copying theinformation encoded in the genome, inthis case by an RNA polymerase that

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Current Biology

Figure 1. Model for how Mrc1 prevents transcription–replication collisions.

In response to osmotic stress, many genes are upregulated by the protein kinase Hog1, poten-tially causing collisions between the transcription and replication machineries. In wild-typecells, Hog1 phosphorylates Mrc1, which in turn inhibits origin firing (initiation) and replisomeprogression (elongation), reducing the probability of replisome and transcription complexcollisions. In mrc13A cells, Mrc1 does not inhibit replication during osmostress, and a DNAdamage response (DDR) results from transcription–replication collisions.

DispatchR201

transcribes one DNA strand into RNA.When a replication and transcriptioncomplex collide, the consequences arepotentially disastrous. In a recentpaper, Duch et al. [2] report an elegantnew mechanism that preventscollisions between replication andtranscription during a heightenedtranscriptional response.

Cells are known to use a variety ofstrategies to resolve the conflictbetween replication and transcription[3,4]. The most dangerous situationinvolves a head-on collision betweenRNA polymerase and the replicationfork. Here, RNA polymerase runsheadlong into the lagging strand DNApolymerase (Figure 1, lower panel). Thefork might be able to displace orcircumvent a single RNA polymerase.However, if it encounters an arrayof RNA polymerases on a highlytranscribed gene, the replication forkcan stall, and eventually collapse,leading to genome rearrangements. InEschericia coli, where the directionalityof replication throughout the genomeis well-defined due to the use ofa single origin, highly transcribedgenes are oriented so as to beco-directional with replication. Anotherstrategy is the use of replication forkbarriers. In the heavily transcribedrDNA locus in eukaryotes, aunidirectional replication fork barrierresiding at the 3’ end of the rDNA geneprevents head-on collisions of RNApolymerase and the replisome.

A problematic situation arises whenthere is a sudden and dramaticincrease in the expression of manygenes, as seen during osmotic stress.In yeast, w600 genes are upregulatedin response to ‘osmostress’ by thestress-activated protein kinase (SAPK)Hog1 [5,6]. Hog1 activates geneexpression by phosphorylatingtranscription factors such as Smp1and Sko1, and by recruiting RNApolymerase and chromatin remodelers[7]. Importantly, Hog1 is activated inresponse to osmotic stress in all stagesof the cell cycle, including S phase [8].How cells manage the conflict betweenreplication and many highly inducedgenes has been unclear.

Duch et al. [2] have tackled thisproblem. In a proteomic screen,they identified the replication factorMrc1 as a direct target of Hog1phosphorylation. Mrc1 is required foroptimal replication fork progression[9–11], possibly due to a physicalinteraction with DNA polymerase

epsilon (Pol ε) [12]. Duch et al. showthat Hog1 phosphorylates Mrc1 onthree MAPK consensus sites. Whenyeast cells experience osmostress,S phase progression is severelydelayed. This effect does not requiresignaling by the Rad53 or Mec1checkpoint kinases. Instead, itdepends critically on Hog1-dependentMrc1 phosphorylation as no S phaseslowing is seen with an MRC1 allele(mrc13A) lacking the three Hog1phosphorylation sites. Therefore,Hog1-dependent phosphorylation ofMrc1 is required to slow S phaseprogression in response toosmostress.

How does Mrc1 phosphorylationdelay S phase? Chromatinimmunoprecipitation (ChIP), 2-D gelelectrophoresis, and DNA-combingassays suggest that the rate ofreplication fork progression issignificantly diminished in response toosmostress, perhaps due to a reducedinteraction between Mrc1 and Pol ε(Figure 1, upper panel). In addition,firing of both early and late origins wasinhibited due to defective loading of thehelicase co-factor Cdc45. The effectsof osmostress on initiation andelongation were abrogated in themrc13A mutant (Figure 1, lower panel),consistent with the lack of a delayedS phase in these cells and with both

effects being mediated throughinhibition of mrc1 function.Duch et al. next addressed the

consequences of disrupting theosmoregulation of Mrc1. They foundthat unlike wild-type (WT) cells, mrc13A

cells exhibited a dramatic increase inchromosomal instability after osmoticshock. They suspected that theobserved chromosomal instabilitymight be due to conflicts between DNAreplication and transcription. To testthis, they used a plasmid in which anosmostress-driven reporter geneconsisting of tandem repeats is locatedin two orientations with respect to anorigin of replication. In the presence offork collapse, the tandem repeatsrecombine, yielding a functionalreporter gene. As such, the systemreads out transcription-associatedrecombination (TAR). Strikingly,mrc13A but not WT cells exhibiteda massive increase in TAR afterosmostress, but only when thepromoter and origin were oriented suchas to yield head-on collisions of thereplication and transcription machines.No TAR was observed withco-directional promoters. Althoughmrc13A cells had normal viability duringosmostress, when they also lackedRad53, viability was reduced. Thissuggests that when the Hog1–Mrc1pathway is disrupted, the damage

Current Biology Vol 23 No 5R202

caused by fork collisions can be dealtwith by the traditional DNA damagecheckpoint. In summary, the dataprovide compelling evidence that cellsattenuate DNA replication duringperiods of heightened transcription toavoid genomic catastrophes.

Mrc1 is not only required forreplication fork progression but also foramplification of checkpoint signalingduring replicative stress, for examplewhen deoxyribonucleotides aredepleted by hydroxyurea (HU) [9,13].Specifically, Mrc1 serves to amplifycheckpoint signaling by the Rad53kinase, which involves Mrc1phosphorylation by Rad53.Importantly, the Rad53 and Hog1phosphorylation sites in Mrc1 aredistinct, and themrc13A allele supportsnormal checkpoint signaling and cellsurvival during replication stress.Thus, Mrc1 participates in distinctosmostress and replication stresspathways, governed by Hog1 andRad53, respectively.

The study raises numerousinteresting questions. One concernsthe mechanism of how Mrc1phosphorylation prevents replicationinitiation. Previous results suggest thatassembly of the Cdc45, Mcm2-7, GINS(CMG) replicative helicase complex isrequired for recruitment ofMrc1andPolε to the replisome [14,15], whereasMrc1 is not normally required forrecruitment of Cdc45 [16]. In this light, itis surprising that Hog1-phosphorylatedMrc1 binds origins and delays loadingof Cdc45 and Pol ε [2]. The data implythat osmostress converts Mrc1 intoa dominant negative inhibitor that bindspre-RCs andprevents loading of Cdc45and Pol ε. A possible precedent forMrc1 functioning as an inhibitor isseen in fission yeast, where deletion ofMrc1 enhances replication initiationefficiency at some origins and whereMrc1 can bind origins independentlyof Cdc45 [17]. In the future, it will beinteresting to explore the mechanisticbasis of how Mrc1 inhibits originfiring under different conditions. Aninteresting point is that slowing downreplication forks should increase ratherthan decrease the probability thatan RNA polymerase encountersa replication fork. Perhaps Mrc1phosphorylation not only slows, butalso stabilizes the fork in the event ofcollision with RNA polymerase.

Another pressing question iswhetherthe conclusions of this study apply tometazoans. In support of this notion,

high osmolarity and other stressespromote phosphorylation of theMCM2-7 loading factor Cdt1 by themammalian SAPKs p38 and JNK,thereby inhibiting origin licensing[18,19]. Whether post-licensing eventsof origin firing and fork progression arealso inhibited, and whether this avoidsclashes with transcription, remains tobe tested. Based on the findings byDuch et al., it seems likely that cellswill use many creative strategies tomanage the conflict betweenreplication and transcription.

References1. Masai, H., Matsumoto, S., You, Z., Yoshizawa-

Sugata, N., and Oda, M. (2010). Eukaryoticchromosome DNA replication: where, when,and how? Annu. Rev. Biochem. 79, 89–130.

2. Duch, A., Felipe-Abrio, I., Barroso, S.,Yaakov, G., Garcia-Rubio, M., Aguilera, A., deNadal, E., and Posas, F. (2013). Coordinatedcontrol of replication and transcription bya SAPK protects genomic integrity. Nature 493,116–119.

3. Bermejo, R., Lai, M.S., and Foiani, M. (2012).Preventing replication stress to maintaingenome stability: resolving conflicts betweenreplication and transcription. Mol. Cell 45,710–718.

4. Pomerantz, R.T., and O’Donnell, M. (2010).What happens when replication andtranscription complexes collide? Cell Cycle 9,2537–2543.

5. O’Rourke, S.M., and Herskowitz, I. (2004).Unique and redundant roles for HOG MAPKpathway components as revealed bywhole-genome expression analysis. Mol. Biol.Cell 15, 532–542.

6. Westfall, P.J., Ballon, D.R., and Thorner, J.(2004). When the stress of your environmentmakes you go HOG wild. Science 306,1511–1512.

7. Saito, H., and Posas, F. (2012). Response tohyperosmotic stress. Genetics 192, 289–318.

8. Yaakov, G., Duch, A., Garcia-Rubio, M.,Clotet, J., Jimenez, J., Aguilera, A., andPosas, F.(2009). The stress-activatedprotein kinaseHog1mediates S phase delay in response toosmostress. Mol. Biol. Cell 20, 3572–3582.

9. Osborn, A.J., and Elledge, S.J. (2003). Mrc1is a replication fork component whosephosphorylation in response to DNA replicationstress activates Rad53. Genes Dev. 17,1755–1767.

10. Szyjka, S.J., Viggiani, C.J., and Aparicio, O.M.(2005). Mrc1 is required for normal progressionof replication forks throughout chromatin inS. cerevisiae. Mol. Cell 19, 691–697.

11. Tourriere, H., Versini, G., Cordon-Preciado, V.,Alabert, C., and Pasero, P. (2005). Mrc1 andTof1 promote replication fork progression andrecovery independently of Rad53. Mol. Cell 19,699–706.

12. Lou, H., Komata, M., Katou, Y., Guan, Z.,Reis, C.C., Budd, M., Shirahige, K., andCampbell, J.L. (2008). Mrc1 and DNApolymerase epsilon function together in linkingDNA replication and the S phase checkpoint.Mol. Cell 32, 106–117.

13. Alcasabas,A.A.,Osborn,A.J.,Bachant, J.,Hu,F.,Werler, P.J., Bousset, K., Furuya, K., Diffley, J.F.,Carr, A.M., and Elledge, S.J. (2001). Mrc1transduces signals of DNA replication stress toactivate Rad53. Nat. Cell Biol. 3, 958–965.

14. Gambus, A., Jones, R.C., Sanchez-Diaz, A.,Kanemaki, M., van Deursen, F.,Edmondson, R.D., and Labib, K. (2006). GINSmaintains association of Cdc45 with MCM inreplisome progression complexes at eukaryoticDNA replication forks. Nat. Cell Biol. 8, 358–366.

15. Pai, C.C., Garcia, I., Wang, S.W., Cotterill, S.,Macneill, S.A., and Kearsey, S.E. (2009). GINSinactivation phenotypes reveal two pathwaysfor chromatin association of replicative alphaand epsilon DNA polymerases in fission yeast.Mol. Biol. Cell 20, 1213–1222.

16. Calzada, A., Hodgson, B., Kanemaki, M.,Bueno, A., and Labib, K. (2005). Molecularanatomy and regulation of a stable replisome ata paused eukaryotic DNA replication fork.Genes Dev. 19, 1905–1919.

17. Hayano, M., Kanoh, Y., Matsumoto, S., andMasai, H. (2011). Mrc1 marks early-firing originsand coordinates timing and efficiency ofinitiation in fission yeast. Mol. Cell. Biol. 31,2380–2391.

18. Chandrasekaran, S., Tan, T.X., Hall, J.R., andCook, J.G. (2011). Stress-stimulatedmitogen-activated protein kinases control thestability and activity of the Cdt1 DNA replicationlicensing factor. Mol. Cell. Biol. 31, 4405–4416.

19. Miotto, B., and Struhl, K. (2011). JNK1phosphorylation of Cdt1 inhibits recruitment ofHBO1 histone acetylase and blocks replicationlicensing in response to stress. Mol. Cell 44,62–71.

Department of Biological Chemistry andMolecular Pharmacology, Harvard MedicalSchool, 240 Longwood Ave, Boston,MA 02115, USA.E-mail: johannes_walter@hms.harvard.edu

http://dx.doi.org/10.1016/j.cub.2013.01.054

Causality: Perceiving the Causes ofVisual Events

Adapting to visual collisions increases the tendency to see the colliding objectsas sliding over one another, rather than as one ‘launching’ another, but only inthe adapted retinal location. This demonstrates a low-level perceptualcomponent to the interpretation of the causes of visual events.

Alan Johnston

Imagine a billiard ball rolling directlytowards another: it makes contact,

stops and then the other ball rollsforwards. Naturally, we see a collisionand have the impression that the firstball caused the second to move.