PROGRESS Pathways of mammalian replication fork restart Eva Petermann and Thomas Helleday
Abstract | Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation. The faithful and complete replication of chromosomes is essential to maintain genetic stability and prevent the accumulation of cancer-promoting mutations. Replication forks are vulnerable to stalling or collapse as they encounter obstacles on the DNA template, which can be unrepaired DNA damage, DNA-bound proteins or secondary structures. Similarly, chemical agents like hydroxyurea and aphidicolin inhibit replication elongation, leading to fork stalling or collapse. A stalled replication fork is arrested but is capable of resuming replication (replication fork restart) once the inhibition is removed, whereas a collapsed fork has become inactivated through dissociation of the replication machinery or the generation of DNA double-strand breaks (DSBs). In eukaryotic cells, fork-associated DSBs are generated by the structurespecific endonuclease complex of methyl methanesulphonate (MMS) and ultravioletsensitive 81 (MUS81) and essential meiotic endonuclease 1 (EME1)1. After the removal of replication inhibitors such as aphidicolin and hydroxyurea, replication resumes by mechanisms that have been poorly understood in mammalian cells. More is known about replication restart in Escherichia coli, in which stalled or collapsed forks are reactivated by recombination-dependent or recombinationindependent pathways, depending on their structure2 (Supplementary information S1 (box)). The proteins involved in these pathways are not conserved in eukaryotes. E. coli
replication is dependent on fork reactivation as bacterial chromosomes and plasmids contain only one replication origin and forks cannot move beyond a specific termination site. By contrast, eukaryotic chromosomes contain more replication origins than are activated during each round of replication and these can serve as backups for completion of replication if progression of nearby forks is impaired3,4. It has therefore been unclear whether eukaryotes use specialized mechanisms for replication restart after the removal of replication inhibitors. Although it has been established that replication inhibitors activate homologous recombination5,6, analysing replication restart has been difficult owing to the lack of methods for studying individual replication forks. However, in recent years, single-molecule analyses of replication using chromosome combing or the DNA fibre technique7 have led to a better understanding of mammalian replication restart8–13. Various proteins that are not part of the core replication machinery promote efficient replication fork restart after the removal of replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. This Progress article focuses on possible restart mechanisms for mammalian replication forks that have been stalled by hydroxyurea and aphidicolin, replication inhibitors that do not seem to modify the DNA template. Broadly, three fork stabilization and restart pathways can be postulated (FIG. 1). In the first mechanism, a stalled fork structure
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might undergo remodelling before restart, such as the re-annealing of excess singlestrand DNA (ssDNA) or fork regression and pairing of the newly synthesized strands to form a Holliday junction in a structure termed a ‘chicken foot’ (FIG. 1a). This fork remodelling would involve fork collapse by the dissociation of the replication machinery but could theoretically be reversed. In a second mechanism, the Holliday junction is used to facilitate fork restart by recombining into the template by strand invasion to form a displacement loop (D-loop; FIG. 1b). In a D‑loop, one strand of a double-stranded DNA molecule is displaced by a third strand of homologous sequence; in this case, a newly synthesized daughter strand. In E. coli, this allows origin-independent loading of the replication machinery 14. In the third mechanism, the Holliday junction is processed into a one-ended DSB. Fork restart is achieved through homologous recombination-mediated repair of the DSB in a mechanism analogous to break-induced replication (BIR)15 (FIG. 1c). Here, we discuss recent evidence, obtained using single-molecule analyses in mammalian cells, for and against each of these fork restart mechanisms. As the ability to analyse replication forks in mammalian cells is a recent advance in the field, we do not cover evidence from non-mammalian model systems. Furthermore, owing to different underlying concepts, we do not cover fork progression on DNA templates modified by DNA-damaging agents. It is important to keep in mind that single-molecule approaches directly measure fork progression and cannot distinguish defects in fork stabilization, which could underlie a restart defect (see Supplementary information S2 (box)). The pathways described below may therefore be active during the replication block to stabilize forks and after the removal of the inhibitor to restart forks. Restart by fork remodelling DNA helicases may promote restart by remodelling and stabilizing stalled fork structures (FIG. 1a). Fork regression into Holliday junctions has not been shown in mammalian cells, but there is no direct evidence against this model. Such a process VOLUME 11 | O CTOBER 2010 | 683
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PROGRESS a
5′ 3′ 5′ 3′
Polymerase–helicase uncoupling 5′ 3′
Restart
ssDNA annealing
Reversal or degradation
5′ 3′
Chicken foot Fork regression
b
D-loop
Double Holliday junction
D-loop formation Restart
c Holliday junction cleavage
One-ended DSB
D-loop
D-loop formation
Figure 1 | Models of replication fork restart. a | Restart by fork remodelling. A stalled replication fork might be stabilized by the re-annealing of single-strand DNA (ssDNA) generated by excessive unwinding of the template, or might undergo regression and pairing of the newly synthesized strands to form a Holliday junction in a structure termed a ‘chicken foot’. Restart after Holliday junction formation may be difficult if it requires the removal and subsequent re-loading of the replication machinery. b | Holliday junction‑mediated fork restart. The double-stranded DNA (dsDNA) end of the Holliday junction is recombined into the template through strand invasion, forming a displacement loop (D‑loop). In E. coli, the D‑loop allows re-loading of the replication machinery. The invading strand re-anneals with
may be beneficial (by stabilizing stalled forks) or detrimental to restart, and both possibilities have to be considered. Three non-replicative helicases are known to promote fork restart after short to mediumlength periods (14 hours) of replication inhibition. BLM facilitates fork restart. The DNA helicase Bloom syndrome helicase (BLM) promotes efficient replication fork restart after 2–6 hours of aphidicolin or hydroxy urea treatment through its helicase activity 8. Reduced fork restart in BLM-deficient cells is accompanied by increased origin firing, suggesting a novel role of BLM in controlling replication initiation8. BLM might stabilize forks either by facilitating fork regression into a Holliday junction or by reversing fork regression through its Holliday junction migration activity 16,17. Additionally, BLM could promote fork restart by repressing the aberrant formation of recombination intermediates at stalled forks18. BLM-deficient cells treated with
Holliday junction dissolution
Restart
Holliday junction resolution
the template, forming a double Holliday junction that can be removed by NaturetoReviews | Molecular exchanges, Cell Biology Holliday junction resolution, which leads sister chromatid or Holliday junction dissolution, which avoids the generation of recombination products42. c | Double-strand break (DSB)-mediated restart. The Holliday junction is processed into a one-ended DSB and fork restart is achieved through homologous recombination repair of the DSB in a mechanism analogous to break-induced replication. The resulting single Holliday junction would be resolved by Holliday junction resolution. The leading strand and leading-strand template are shown in light and dark blue, respectively, and the lagging strand and lagging-strand template are shown in light and dark red, respectively.
replication inhibitors display nuclear foci of the central homologous recombination factor RAD51, which is a sign of increased homologous recombination initiation19. This could be interpreted as elevated, unscheduled recombination, but also as an increased need for the repair of forks collapsed into DSBs or a failure to complete homologous recombination. WRN enables fork progression during restart. Like BLM, Werner syndrome ATPdependent helicase (WRN) can catalyse both fork regression and Holliday junction migration in vitro20,21. WRN depletion in fibroblasts does not affect the percentage of forks restarting after release from short (4–7 hour) hydroxyurea treatment; instead, the speed of the restarting forks is reduced10. This suggests that WRN is not needed to stabilize forks but is required for efficient fork progression during restart. In WRNdepleted HeLa cells, however, fewer forks restart after 6–14 hours of hydroxyurea treatment, indicating fork inactivation9.
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This discrepancy may not reflect a specific feature of WRN, but could be due to the different time points used or different kinetics of fork inactivation in these cells. In fibroblasts, WRN also promotes fork progression after MMS treatment 10, suggesting that WRN acts on structures such as single-strand gaps left behind the fork. This prompted the speculation that WRN remodels forks to coordinate fork progression with repair of gaps or lesions10,22. ssDNA annealing promotes fork restart. SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A‑like 1 (SMARCAL1) is a ssDNA annealing helicase that is mutated in Schimke immunoosseous dysplasia23 and was recently implicated in the response to replication inhibitors. SMARCAL1 is recruited to stalled replication forks or sites of DNA damage containing ssDNA12,24. Cells depleted of SMARCAL1 accumulate ssDNA and DNA damage and display increased sensitivity to aphidicolin and hydroxyurea12,24. www.nature.com/reviews/molcellbio
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PROGRESS DNA fibre analyses show that replication fork restart after 2 hours of aphidicolin treatment is reduced in SMARCAL1‑depleted cells12. These data suggest that SMARCAL1 promotes fork stability and restart by reannealing long stretches of ssDNA generated at stalled replication forks.
Glossary Aphidicolin
Holliday junction
A small-molecule inhibitor that directly blocks the activity of the replicative DNA polymerases. Aphidicolin treatment leads to replication fork stalling and eventually DNA DSB formation.
A four-way junction between two dsDNA molecules of homologous sequence. Holliday junctions are mobile and can be translocated by DNA helicases (branch migration).
Hydroxyurea Break-induced replication
Considerations against restart by remodelling. The main conceptual weakness of fork restart by remodelling is that the replication machinery presumably needs to dissociate from the replication fork to allow remodelling and it is not clear how it would be re-loaded. All fork remodelling might therefore channel forks into the Holliday junction‑mediated or DSB-mediated restart pathways.
A mechanism for origin-independent replication restart, whereby a resected DNA end invades a homologous DNA molecule, thus establishing a replication fork.
Holliday junction-mediated restart The idea that fork restart can be achieved by a Holliday junction intermediate without DSB formation is mainly supported by the observation that fork restart requires the recombination factor RAD51 (which catalyses Holliday junction formation) but not necessarily MUS81 (which cleaves Holliday junctions into one-ended DSBs)9,13. RAD51‑mediated restart is seen after short (2 hour) but not long (24 hour) hydroxyurea treatments13. RAD51 is loaded onto ssDNA to form protein–DNA filaments, which then catalyse homology search and strand exchange25, activities that would be required for strand invasion and D‑loop formation during Holliday junction‑mediated restart. As regression into a Holliday junction prob ably requires dissociation of the replication machinery, this restart pathway may act on forks that have collapsed by losing most of their replication factors.
Core replication machinery
Generation of the Holliday junction. Both BLM and WRN could facilitate fork regression into a Holliday junction through their helicase activities17,21. Another way of generating a Holliday junction could be through RAD51. The bacterial RAD51 homologue, RecA, can convert specific types of stalled forks into a Holliday junction by pairing the template strands26,27, and RAD51 can facilitate a similar mechanism28. Requirement of DNA end processing. RAD51 loading onto DNA requires a lagging-strand gap or a 3′-overhang and, therefore, probably involves end processing. These structures may be generated by the exonuclease activity of meiotic recombination 11 (MRE11) as MRE11‑dependent DNA resection has been shown to promote
A radical scavenger that inhibits ribonucleotide reductase, which results in cells producing less of the desoxyribo nucleotides that are used for DNA synthesis. Hydroxyurea treatment leads to replication fork stalling and eventually DNA DSB formation.
Chromosome combing A method for single-molecule analysis of DNA replication forks. Newly replicated DNA is labelled in vivo using halogenated thymidine analogues and genomic DNA is isolated and spread out on microscope coverslips. Immunofluorescence staining of the thymidine analogues is used to visualize the labelled tracks that are left on the DNA by moving replication forks.
The complex of proteins that is essential for all DNA replication and includes the replicative DNA helicase, primase, clamp loader, sliding clamp and leading- and lagging-strand DNA polymerases.
Lagging strand The nascent strand of DNA that is synthesized discontinuously in short pieces (Okazaki fragments) at the replication fork.
Origin firing The start of replication fork progression at a replication origin. Mammalian origin firing is restricted to S phase and is controlled by cell cycle signalling.
Replication fork A structure formed when the template strands have been separated by helicases and a newly formed copy of the DNA is synthesized. The fork moves in the direction of leading-strand synthesis.
DNA fibre technique A technique that is similar to chromosome combing, but in which cells on microscope slides are treated with detergent and the DNA is spread directly out of the lysed nuclei. Because of the lysis method, this technique is used in vertebrate cells but not in yeast.
Replication fork restart The resumption of fork progression after removal or bypass of a replication block.
Replication fork stabilization The maintenance of viable replication fork structures and the replication machinery during a replication block.
DNA helicase An enzyme that translocates on DNA and unwinds the double helix into ssDNA in an ATP-driven reaction. Annealing helicases use ATP to catalyse the reverse reaction.
homologous recombination29,30. Supporting this, DNA fibre analyses show that MRE11 facilitates replication fork restart after release from a short hydroxyurea-mediated block11 acting in the same pathway as the DNA damage signalling protein poly(ADPribose) polymerase 1 (PARP1). PARP1 seems to recruit MRE11 to facilitate fork restart by the resection of DNA ends11. BLM and WRN might function in the same pathway, as both physically or functionally interact with MRE11 (REFS 31,32). BLM promotes MRE11 foci formation in response to hydroxyurea31. Interestingly, WRN interacts with MRE11 during short (4 hour), but not longer (8 hour) hydroxyurea treatments, suggesting that the role of this interaction is specific to short replication blocks32. Observations from budding yeast suggest that MRE11 might alternatively or additionally promote fork restart by facilitating sister chromatid cohesion33, which also regulates normal replication fork progression in human cells34.
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Replication origin The chromosomal location at which new replication factories are assembled and replication is initiated. Replication fork movement from origins is bidirectional.
RAD51 loading and strand invasion. The RAD51 paralogue X‑ray repair crosscomplementing protein 3 (XRCC3), which mediates RAD51 filament formation35, also promotes fork restart after a short hydroxyurea-mediated block 13. This implicates RAD51‑mediated filament formation in the restart process. Nevertheless, RAD51‑mediated fork restart does not involve RAD51 foci formation or detectable, long patch recombination in a reporter construct 13. This suggests that the extent of RAD51 loading and the recombination mechanism differ from homologous recombination‑mediated DSB repair. Visible foci would require numerous RAD51 molecules to be present at the forks in, for example, long filaments. Detectable recombination at a distant site might be prevented in the absence of a DSB at restarting forks. Fork restart that does not generate recombination products would be supported by BLM, which, in a complex with DNA topoisomerase IIIα (TOPIIIα), resolves double VOLUME 11 | O CTOBER 2010 | 685
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PROGRESS Hydroxyurea-mediated block (S phase)
Length of hydroxyurea treatment
0 hours
Stalled fork 5′ 3′ 5′ 3′ MUS81
Release from block (S phase)
SMARCAL1 BLM WRN PARP1 MRE11 XRCC3 RAD51
Fork restart
Completed replication
5′ 3′
5′ 3′
5′ 3′
5′ 3′
5′ 3′
5′ 3′ Collapsed fork 24 hours
Late S–G2 phase
New origin firing
PARP1 MRE11 XRCC3 RAD51 BLM WRN
5′ 3′ 5′ 3′ Homologous recombinationmediated repair
Figure 2 | Early replication fork restart and late replication fork repair. Most replication forks Nature Reviews | Molecular Cell Biology resume progression after release from short (2–4 hour) hydroxyurea-mediated blocks. Restart is promoted by the DNA helicases Bloom syndrome helicase (BLM) and Werner syndrome ATP-dependent helicase (WRN), the annealing helicase SWI/SNF-related matrix-associated actin-dependent regu lator of chromatin subfamily A-like protein 1 (SMARCAL1), the DNA damage signalling protein poly (ADP-ribose) polymerase 1 (PARP1; which recruits the nuclease and double-strand break (DSB) repair protein meiotic recombination 11 (MRE11)), and the homologous recombination proteins X-ray repair cross-complementing protein 3 (XRCC3) and RAD51. Stalled forks become inactivated during long (24 hour) hydroxyurea treatments. Within one hour after release from these long blocks, replication restarts by the firing of new origins. Fork inactivation coincides with the accumulation of DSBs that are dependent on the structure-specific endonuclease MMS and UV-sensitive protein 81 (MUS81). DSB formation does not seem to promote restart but does promote the RAD51‑dependent, postreplicative recombination repair of forks, shown here as synthesis-dependent strand annealing. PARP1, MRE11, BLM and WRN promote DSB repair. Hydroxyurea ‑induced DSBs can persist for more than 48 hours. Thus, after short hydroxyurea treatments, DNA replication resumes mainly by fork restart, whereas after long hydroxyurea treatments, replication restarts by the firing of new origins, followed by DSB repair of collapsed forks.
Holliday junctions in a process that avoids crossing over 36–38. DSB-mediated restart If mammalian replication forks are stalled for many hours, fork-associated DSBs are generated by MUS81–EME1 (REFS 1,6) (FIG. 1c), suggesting that DSB formation may play a part in replication fork restart, especially after long periods of replication inhibition.
Evidence for DSB-mediated fork restart. MUS81‑deficient mouse embryonic stem cells display a fork restart defect, especially after 24 hours of hydroxyurea treatment 1. This suggests that DSB generation does indeed promote fork restart. Interestingly, during treatments with low doses of aphidicolin that do not completely block replication, BLM and MUS81 facilitate a transient surge of DSBs during the first hour of treatment, and promote residual replication fork progression during that time39. This suggests that DSB-mediated fork restart can promote replication fork progression in the presence of low levels of a replication inhibitor.
Evidence against DSB-mediated fork restart. Other recent evidence suggests that DSB generation does not play a part in fork restart in human cancer cells. Although most stalled replication forks in human U2OS cells resume progression after short hydroxyurea treatments, they do not restart after long hydroxyurea treatments13 (FIG. 2). Furthermore, small interfering RNA depletion of MUS81 has no effect on fork restart in HeLa and U2OS cells (REF. 9; E.P and T.H, unpublished observations). These observations suggest that DSB formation at stalled forks is not necessarily part of a restart process. In Saccharomyces cerevisiae, initiation of BIR, a mechanism equivalent to DSB-mediated restart, takes several hours40. A similar delay could account for the observation that in U2OS cells, replication restarts by a new origin firing within one hour of being released from a long hydroxyureamediated block13. The conflicting reports described here could be due to different cell types and organisms used. In some rodent cells, increased fork inactivation could channel forks into DSB-mediated restart. Mouse embryonic stem cells and Chinese hamster ovary (CHO) cells display earlier
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hydroxyurea‑induced RAD51 foci formation than human cells, and in CHO cells this is accompanied by increased fork inactivation1,13. WRN-deficient cells also display early fork inactivation and require MUS81 for efficient S phase progression after release from a hydroxyurea-mediated block, suggesting that MUS81 contributes to replication restart in these cells9. Fork inactivation and repair. Although inactivation of stalled replication forks coincides with the accumulation of DSBs, DSB formation does not seem to be the cause of fork inactivation as MUS81 depletion does not improve fork restart after long hydroxyurea treatments13. WRN-deficient cells display increased MUS81 activity, but MUS81 co-depletion does not rescue their fork restart defect 9. Instead, MUS81 promotes homologous recombination-mediated repair of the inactivated forks9. This suggests that fork inactivation might be caused by the dissociation of replication proteins and possibly fork remodelling. Fork cleavage by MUS81 is a result of fork inactivation and is a prerequisite for DSB-mediated fork repair, a process that also requires RAD51 (REF. 13). Other proteins that promote early fork restart (BLM, WRN, PARP1 and MRE11) also have roles in homologous recombination11,16,20,29,30 and may be involved in fork repair, depending on the length of replication inhibition. With most stalled forks inactivated after long hydroxyurea treatments, global replication restart is achieved by new origin firing 13. If homologous recombination (and possibly non-homologous end joining) can repair the vast number of DSBs, then genomic instability may be prevented. Conclusions The findings described here have helped to increase our understanding of mammalian replication fork restart. The proposed pathways are required for efficient restart of a subset of replication forks, suggesting that most forks are able to restart directly. A notable exception is the SET domain and mariner transposase fusion gene-containing protein (METNASE; also known as SETMAR), a primate-specific methyltransferase and nuclease that is required for the restart of most forks after hydroxyurea treatment 41,42. However, replication initiation is also suppressed in METNASE-depleted cells, suggesting that there is a general block to replication rather than a specific fork restart defect 42. Replication forks requiring special pathways for reactivation may have undergone additional structural changes. www.nature.com/reviews/molcellbio
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PROGRESS In such cases, remodelling of stalled fork structures by DNA helicases and homologous recombination factors promotes the efficient resumption of fork progression. DSB formation is not necessarily a first step in fork restart but can be a result of fork inactivation. Homologous recombination is activated for the post-replicative repair of collapsed forks. However, many aspects remain to be investigated. The molecular details of replication fork stabilization and Holliday junction‑mediated restart are not yet clear. Analysing the requirement for homologous recombination proteins that promote different steps during Holliday junction‑mediated restart will help elucidate the molecular mechanism of this process. Although most proteins mentioned are known to promote homologous recombination, it is currently unknown whether they act in the same restart pathway and subset of replication forks. It is also unclear how replication complexes are re-loaded at remodelled replication forks. Other pathways, such as checkpoint signalling 43–45 and sister chromatid cohesion46,47, stabilize stalled replication forks and may also promote fork restart (Supplementary information S3 (box)). Global inhibitors of replication, such as hydroxyurea and aphidicolin, differ from more physiological, stochastic inhibitors of individual forks in that they are more likely to activate cell cycle checkpoint signalling, which will affect the processing of stalled forks. It will therefore be interesting to develop methods to study rare fork‑stalling events. Inaccurate replication fork restart may be an important source of genomic instability. In the future, single-molecule analyses of DNA replication will become increasingly important for addressing this issue in mammalian cells. Eva Petermann is at the School of Cancer Sciences, University of Birmingham, Birmingham, B15 2TT, UK. Thomas Helleday is at the Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, OX3 7DQ, UK, and at the Department of Genetics Microbiology and Toxicology, Stockholm University, S‑106 91 Stockholm, Sweden.
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[email protected] doi:10.1038/nrm2974 Published online 15 September 2010 1.
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licensing backup origins of replication. Proc. Natl Acad. Sci. USA 105, 8956–8961 (2008). Ge, X. Q., Jackson, D. A. & Blow, J. J. Dormant origins licensed by excess MCM2–7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341 (2007). Lundin, C. et al. Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells. Mol. Cell. Biol. 22, 5869–5878 (2002). Saintigny, Y. et al. Characterization of homologous recombination induced by replication inhibition in mammalian cells. EMBO J. 20, 3861–3870 (2001). Tuduri, S., Tourriere, H. & Pasero, P. Defining replication origin efficiency using DNA fiber assays. Chromosome Res. 18, 91–102 (2010). Davies, S. L., North, P. S. & Hickson, I. D. Role for BLM in replication-fork restart and suppression of origin firing after replicative stress. Nature Struct. Mol. Biol. 14, 677–679 (2007). Franchitto, A. et al. Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81‑dependent pathway. J. Cell Biol. 183, 241–252 (2008). Sidorova, J. M., Li, N., Folch, A. & Monnat, R. J. Jr. The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest. Cell Cycle 7, 796–807 (2008). Bryant, H. E. et al. PARP is activated at stalled forks to mediate Mre11‑dependent replication restart and recombination. EMBO J. 28, 2601–2615 (2009). Ciccia, A. et al. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart. Genes Dev. 23, 2415–2425 (2009). Petermann, E., Orta, M. L., Issaeva, N., Schultz, N. & Helleday, T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51‑mediated pathways for restart and repair. Mol. Cell 37, 492–502 (2010). Liu, J., Xu, L., Sandler, S. J. & Marians, K. J. Replication fork assembly at recombination intermediates is required for bacterial growth. Proc. Natl Acad. Sci. USA 96, 3552–3555 (1999). Llorente, B., Smith, C. E. & Symington, L. S. Breakinduced replication: what is it and what is it for? Cell Cycle 7, 859–864 (2008). Karow, J. K., Constantinou, A., Li, J. L., West, S. C. & Hickson, I. D. The Bloom’s syndrome gene product promotes branch migration of holliday junctions. Proc. Natl Acad. Sci. USA 97, 6504–6508 (2000). Ralf, C., Hickson, I. D. & Wu, L. The Bloom’s syndrome helicase can promote the regression of a model replication fork. J. Biol. Chem. 281, 22839–22846 (2006). Bugreev, D. V., Yu, X., Egelman, E. H. & Mazin, A. V. Novel pro- and anti-recombination activities of the Bloom’s syndrome helicase. Genes Dev. 21, 3085–3094 (2007). Rassool, F. V., North, P. S., Mufti, G. J. & Hickson, I. D. Constitutive DNA damage is linked to DNA replication abnormalities in Bloom’s syndrome cells. Oncogene 22, 8749–8757 (2003). Constantinou, A. et al. Werner’s syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Rep. 1, 80–84 (2000). Machwe, A., Xiao, L., Groden, J. & Orren, D. K. The Werner and Bloom syndrome proteins catalyze regression of a model replication fork. Biochemistry 45, 13939–13946 (2006). Sidorova, J. M. Roles of the Werner syndrome RecQ helicase in DNA replication. DNA Repair (Amst.) 7, 1776–1786 (2008). Yusufzai, T. & Kadonaga, J. T. HARP is an ATP-driven annealing helicase. Science 322, 748–750 (2008). Bansbach, C. E., Betous, R., Lovejoy, C. A., Glick, G. G. & Cortez, D. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks. Genes Dev. 23, 2405–2414 (2009). Baumann, P., Benson, F. E. & West, S. C. Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766 (1996). Seigneur, M., Ehrlich, S. D. & Michel, B. RuvABCdependent double-strand breaks in DnaBts mutants require RecA. Mol. Microbiol. 38, 565–574 (2000). Robu, M. E., Inman, R. B. & Cox, M. M. RecA protein promotes the regression of stalled replication forks in vitro. Proc. Natl Acad. Sci. USA 98, 8211–8218 (2001).
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28. Yoon, D., Wang, Y., Stapleford, K., Wiesmuller, L. & Chen, J. p53 inhibits strand exchange and replication fork regression promoted by human Rad51. J. Mol. Biol. 336, 639–654 (2004). 29. Buis, J. et al. Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation. Cell 135, 85–96 (2008). 30. Sartori, A. A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007). 31. Franchitto, A. & Pichierri, P. Bloom’s syndrome protein is required for correct relocalization of RAD50/ MRE11/NBS1 complex after replication fork arrest. J. Cell Biol. 157, 19–30 (2002). 32. Franchitto, A. & Pichierri, P. Werner syndrome protein and the MRE11 complex are involved in a common pathway of replication fork recovery. Cell Cycle 3, 1331–1339 (2004). 33. Tittel-Elmer, M., Alabert, C., Pasero, P. & Cobb, J. A. The MRX complex stabilizes the replisome independently of the S phase checkpoint during replication stress. EMBO J. 28, 1142–1156 (2009). 34. Terret, M. E., Sherwood, R., Rahman, S., Qin, J. & Jallepalli, P. V. Cohesin acetylation speeds the replication fork. Nature 462, 231–234 (2009). 35. Bishop, D. K. et al. Xrcc3 is required for assembly of Rad51 complexes in vivo. J. Biol. Chem. 273, 21482–21488 (1998). 36. Raynard, S., Bussen, W. & Sung, P. A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIα, and BLAP75. J. Biol. Chem. 281, 13861–13864 (2006). 37. Singh, T. R. et al. BLAP18/RMI2, a novel OB‑fold‑containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome. Genes Dev. 22, 2856–2868 (2008). 38. Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003). 39. Shimura, T. et al. Bloom’s syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress. J. Mol. Biol. 375, 1152–1164 (2008). 40. Malkova, A., Naylor, M. L., Yamaguchi, M., Ira, G. & Haber, J. E. RAD51‑dependent break-induced replication differs in kinetics and checkpoint responses from RAD51‑mediated gene conversion. Mol. Cell Biol. 25, 933–944 (2005). 41. Lee, S. H. et al. The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair. Proc. Natl Acad. Sci. USA 102, 18075–18080 (2005). 42. De Haro, L. P. et al. Metnase promotes restart and repair of stalled and collapsed replication forks. Nucleic Acids Res. 10 May 2010 (doi:10.1093/nar/gkq339). 43. Feijoo, C. et al. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra‑S phase checkpoint monitoring replication origin firing. J. Cell Biol. 154, 913–923 (2001). 44. Zachos, G., Rainey, M. D. & Gillespie, D. A. Chk1‑dependent S‑M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. Mol. Cell Biol. 25, 563–574 (2005). 45. Scorah, J. & McGowan, C. H. Claspin and Chk1 regulate replication fork stability by different mechanisms. Cell Cycle 8, 1036–1043 (2009). 46. Leman, A. R., Noguchi, C., Lee, C. Y. & Noguchi, E. Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion. J. Cell Sci. 123, 660–670 (2010). 47. Tanaka, H. et al. Replisome progression complex links DNA replication to sister chromatid cohesion in Xenopus egg extracts. Genes Cells 14, 949–963 (2009).
Acknowledgements
We thank the Medical Research Council, the Swedish Research Council, the Swedish Children’s Cancer Foundation, the Swedish Pain Relief Foundation and the Swedish Cancer Society for financial support.
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION Thomas Helleday’s homepage: http://helleday.gmt.su.se
SUPPLEMENTARY INFORMATION See online article: S1 (box) | S2 (box) | S3 (box) ALL LINKS ARE ACTIVE IN THE ONLINE PDF
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REVIEWS 1 0 - Y E A R A N N I V E R S A RY S E R I E S
Revitalizing membrane rafts: new tools and insights Kai Simons and Mathias J. Gerl
Abstract | Ten years ago, we wrote a Review on lipid rafts and signalling in the launch issue of Nature Reviews Molecular Cell Biology. At the time, this field was suffering from ambiguous methodology and imprecise nomenclature. Now, new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking. Caveola A 50–80‑nm, flask-shaped pit that forms in the plasma membrane and is enriched in caveolins, cavins, sphingolipids and cholesterol.
Max Planck Institute of Cell Biology and Genetics, Pfotenhauerstraβe 108, 01307 Dresden, Germany. e-mails:
[email protected];
[email protected] doi:10.1038/nrm2977
Cell membranes contain hundreds of lipids in two asymmetric leaflets1 and a plethora of proteins. For several decades, membrane research was dominated by the idea that proteins were the key factors for membrane functionality, whereas lipids were regarded as a passive, fluid solvent 2. Introducing the lipid raft concept in 1997, we postulated that sphingolipid–cholesterol–protein assemblies could function in membrane trafficking and signalling 3. These assemblies, or rafts, were thought to be characterized by their tight lipid packing, similar to the sterol-dependent, liquid-ordered phase in model membranes. The novelty of the raft concept was that it brought lipids back into the picture by giving them a function and by introducing chemical specificity into the lateral heterogeneity of membranes. When we wrote our first Review in this journal4, the emerging raft field had become increasingly confused by ambiguous methodology and imprecise nomenclature. Caveolae, for example, became synonymous with rafts but clearly represented only a subset of membrane assemblies defined by the action of the protein caveolin5. Complicating matters further was the size of the sphingolipid–cholesterol–protein assemblies being studied, which were below the resolution of light microscopy. Only after cross-linking did raft proteins and lipid constituents cluster together to form micrometre-size, quilt-like patches. Our focus in the first Review was to emphasize that rafts are small and dynamic and can be stabilized to form larger microdomains that function in membrane trafficking and signalling. We proposed that three types of assembly should be recognized in cell membranes — rafts, clustered rafts and caveolae (a subset of clustered rafts) — and that the residue remaining insoluble after detergent extraction should be called
detergent-resistant membrane (DRM) fractions. We also summarized the tools that were available for defining rafts and discussed their strengths and shortcomings. Obviously, what was known about lipid rafts and membrane organization at the time was dependent on the available methodology. The rationale of the present Review is to summarize where we stand today and to highlight the important role that new technology has had in moving the field forwards (BOX 1). We describe how membrane rafts are now defined as dynamic, nanoscale, sterol– sphingolipid-enriched, ordered assemblies of proteins and lipids, in which the metastable raft resting state can be stimulated to coalesce into larger, more stable raft domains by specific lipid–lipid, protein–lipid and protein–protein oligomerizing interactions (FIG. 1). The lipids in these assemblies are thought to be enriched in saturated hydrocarbon chains. We describe advances in our understanding of how lipid rafts function as a membrane organizing principle in cellular processes such as T cell signalling, viral infection and membrane trafficking, and also try to identify issues that need to be resolved.
Controversies then and now A key issue ten years ago was the methodology used to define a raft component. An increasing number of papers used detergents as the main criterion for raft association; raft constituents were defined simply as the insoluble residue or DRM remaining after non-ionic detergent solubilization at 4ºC. This criterion was usually combined with the use of methyl‑β-cyclodextrin to extract cholesterol from cell membranes. If the protein became detergent-soluble after cyclodextrin treatment, this strengthened the conclusion that it’s
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REVIEWS Box 1 | Changes in the raft concept Our concept of rafts has shifted with the realization that the association of components is dynamic and sizes range from small, short-lived, nanoscale assemblies to more stable membrane domains, the size and lifetime of which also vary. The apical membranes of epithelial cells, for example, can behave as large percolating raft domains116 or as a super-raft91,117. Similarly, the myelin sheets that oligodendrocytes produce to be wrapped around neuronal axons are another specific raft membrane type118. The situation is like that of logs in a river: one or several logs can function as a raft for one or more loggers and these can pile up into a raft jam. Discussing raft size as a criterion is irrelevant and the dynamics of raft lipids and proteins must be considered. Also, different methods and conditions used to study raft behaviour will give differing results. For example, fluorescence recovery after photobleaching (FRAP) may measure no difference in diffusion rates between raft and non-raft constituents, whereas stimulated emission depletion (STED) microscopy will30 (FIG. 2). The definition of a raft marker has evolved. Protein association with rafts was previously defined by sterol dependency and detergent resistance. Among putative raft lipids, the ganglioside GM1 was a commonly used marker because a fluorescently labelled probe was available. Patching of putative raft constituents was also done by antibody cross-linking in a way that would induce large raft clusters, potentially recruiting every membrane constituent with an affinity for the patched raft membrane environment. Now when nanoscale assemblies are analysed, one does not expect every marker to be enriched, but instead only a limited subset of proteins and lipids16. When these assemblies are clustered into raft platforms, there is no obligatory reason why GPI-anchored proteins, GM1 or other raft constituents should be enriched. Only when larger patches are produced, such as in phase-segregated plasma membrane preparations, would such markers be considered useful.
Förster resonance energy transfer A fluorescence-based method for detecting interactions between fluorophores that are
