S-phase and DNA damage activated establishment of Sister chromatid cohesion—importance for DNA repair

E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 1 4 4 5– 1 45 3

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Review

S-phase and DNA damage activated establishment of Sister chromatid cohesion—importance for DNA repair Camilla Sjögren, Lena Ström⁎ Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden

A R T I C L E I N F O R M A T I O N

AB S TR AC T

Article Chronology:

By holding sister chromatids together from the moment of their formation until their separation at

Received 6 November 2009

anaphase, the multi subunit protein complex Cohesin guarantees correct chromosome

Revised version received

segregation. This S-phase established chromatid cohesion is also essential for repair of DNA

17 December 2009

double strand breaks (DSB) in postreplicative cells. In addition, Cohesin has to be recruited to a

Accepted 21 December 2009

DSB, and new cohesion has to form in response to the damage for repair. When it became clear that

Available online 4 January 2010

cohesion is created de novo in response to DNA breaks, the term “damage induced cohesion” (DI-cohesion) was coined. It is now established that certain factors are needed for establishment of

Keywords:

both S-phase and DI-cohesion, while others have been found to be unique for respective process.

Cohesin

In addition, post-translational modifications of Cohesin components that are functionally

Cohesion

important for cohesion formation, either during S-phase or in response to damage, have

Damage induced cohesion

recently been identified. Here, we present and discuss the current models for establishment of

Cell cycle

S-phase and DI-cohesion in the context of their involvement in DSB repair.

DNA double strand-break

© 2009 Elsevier Inc. All rights reserved.

DNA repair

Contents Introduction . . . . . . . . . . . . . . . . . . The Cohesin complex . . . . . . . . . . . . . Cohesin in DNA repair. . . . . . . . . . . . . Cohesion establishment during S-phase. . . . Regulation of damage induced cohesion . . . How important is DI-cohesion for DSB repair? Acknowledgments . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Box 285, S-171 77 Stockholm. E-mail address: [email protected] (L. Ström). 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.12.018

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Introduction DNA damage is a constant threat to the integrity of the genetic information harboured by every single cell. A DNA double strand break (DSB), where both DNA strands are broken is one of the most deleterious types of damage. Hence correct DSB repair is of outmost importance. In response to DNA damage various checkpoints are activated that halt cell cycle progression and provide time for completion of DNA repair. Depending on cell type and cell cycle phase two different strategies are used for DSB repair; Non Homologous End Joining (NHEJ) and homologous recombination (HR) [1]. NHEJ results in re-ligation of the broken DNA, and frequently leads to loss of genetic information at the break site. HR depends on a homologous DNA template for repair, and is preferentially performed during the S and G2 cell cycle phases, using a sister chromatid as template [2]. Since the sister chromatids are identical copies of each other, this type of repair will leave the genetic information unchanged. After initial recognition of the damage, an early step of HR is DNA resection around the DSB, which creates 3′-OH single-stranded DNA (ssDNA) overhangs to which RPA (replication protein A) binds (Fig. 1). RPA is later replaced by Rad51 in a Rad52-dependent manner, creating a Rad51 coated DNA filament used for homology search and strand invasion. DNA synthesis from the invading ssDNA strand is then initiated, with the homologous region as template. Intermediate recombination structures are finally ligated and resolved (for a review see [3]). Since HR requires close proximity between the broken DNA molecule and the repair template, it was an early prediction that sister chromatid cohesion should have an important role in this process. This could not be directly tested before the protein complex Cohesin was identified as the “glue” that holds the sister chromatids together [4,5].

The Cohesin complex Cohesin forms together with Condensin and the Smc5/6 complex a family of large multi-subunit complexes that are mainly important for correct chromosome segregation, recombinational DNA repair and gene regulation [6,7]. The cores of these complexes are built from Structural Maintenance of Chromosome (SMC) proteins, which are essential for survival and conserved from yeast to man. SMC like proteins are also found in bacteria [8]. In all eukaryotes analyzed, the SMC protein family consists of at least six members, Smc1–6. SMC proteins contain two coiled-coil stretches separated

Fig. 1 – A schematic representation of the homologous recombination pathway, displaying the steps of importance for the understanding of the discussion in this review. A DSB is initially recognized by the Mre11 containing MRX complex. 5′ to 3′ resection of DNA ends is then initiated. Tel1 is recruited to the DSB and Phosphorylation of H2A induced. Single stranded DNA is then bound by RPA, which attracts Mec1, involved in Phosphorylation of H2A and activation of Chk1. Rad51 then replaces RPA in a Rad52 dependant manner. Homology search and strand invasion is executed through the Rad51 presynaptic filament. For further details see text and Ref. [3].

E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 1 4 4 5– 1 45 3

by a central hinge domain, and with globular N and C termini. The proteins fold back on themselves at the hinge domains, bringing together their N and C terminal heads. At their intersection a functional ATPase domain, required for the function of the complexes, is created. Three specific SMC hetero-dimers are formed via interactions through their hinge domains and build the cores of Cohesin (Smc1/3), Condensin (Smc2/4), and the Smc5/6 complex (Smc5/6) [9]. An additional protein with similar structural features as the SMC proteins is Rad50, notably also important for DNA repair [6,10]. In addition to Smc1 and Smc3, the Cohesin complex consists of the non-SMC proteins Scc1 (Mcd1/Rad21) and Scc3 (SA1, 2). Scc1 bridges the N- and C-terminal domains of Smc1 and Smc3, and Scc3 is stably associated with the complex via Scc1 [9]. Furthermore, the accessory proteins Pds5 and Wpl1, which regulate cohesion establishment and maintenance, have in budding yeast been shown to interact with the complex via Scc3 [11]. Cohesin also weakly associates with the Scc2/4 protein complex, needed for Cohesin's chromosomal association [12]. Vast evidence suggests that Smc1 and 3 together with Scc1 physically hold the sisters together through entrapment inside the ringshaped structure formed by these three proteins [9,13]. The ring model is however still under debate and alternative modes for the interaction between DNA and Cohesin, as well as cohesion formation have been suggested [14–16]). At anaphase cohesion has to be released to allow segregation of sister chromatids. This is triggered by the ubiquitin ligase APC (anaphase promoting complex) that induces degradation of the socalled Securin protein. Securin has until then been associated with a protease called Separase, which after Securin degradation is free to cleave its substrate Scc1. This removes Cohesin from chromosomes and allows their segregation into the two new daughter cells [17]. It is important to note that arm cohesion is differently regulated in yeast and human cells. While all Cohesin remain on chromosomes in budding yeast, arm-Cohesin is removed already during prophase in metazoan cells. This removal depends in part on a process regulated by phosphorylation of the human Cohesin subunit Scc3 (SA2) by the Polo like kinase 1 (PLK1) [18–20]. Thus, any cohesion-dependent repair should be completed before this event, and since centromeric cohesion apparently is sufficient for correct chromosome segregation, the main purpose of mammalian arm cohesion could be to promote DNA repair [21]. A schematic representation of the Cohesin complex as well as the modifications and factors required for Cohesin loading and cohesion establishment during the cell cycle are shown in Figs. 2A–B.

Cohesin in DNA repair An early observation of a repair-function for Cohesin was that a mutated version of the Scc1 ortholog in Schizosaccharomyces pombe (Rad21) rendered cells sensitive to γ-irradiation and defective in DSB repair [22]. At this time the function of Scc1/ Rad21 in cohesion was not known. The finding that DNA repair efficiency increases ten thousand fold when yeast cells go from G1 to G2, suggested that completion of replication, i.e. formation of sister chromatids, is important for repair [23]. A formal proof that S-phase cohesion is essential for DSB repair was presented in a study of budding yeast cells. If these underwent replication in the absence of a functional Cohesin complex, DSB repair in G2 was

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severely impaired, and reintroduction of Cohesin in the postreplicative cells could not rescue this deficiency. Thus, proper establishment of cohesion during S-phase is required for repair [5]. The chromosome repair function for Cohesin appears to have been conserved during evolution. The bacterial ancestors to SMC proteins have functions in DNA repair [24]. Furthermore, studies in Scc1-depleted chicken DT40 cells, cell lines from breast cancer patients with impaired Scc1 function, and after RNAi inhibition of Scc1 expression in HeLa cells show that Cohesin is important for DNA repair also in higher eukaryotes [25–27]. In addition to a direct role in repair, Cohesin is also involved in DNA damage checkpoint regulation. Phosphorylation of both hSmc1 and hSmc3 has been shown to be important for induction of the intra S-phase checkpoint in response to DNA damage. Overexpression of Smc1, which cannot be phosphorylated, leads to continuation of DNA synthesis before DNA repair is completed [28–30]. Recently, it was also shown that Cohesin is involved in the damage-induced checkpoint during G2. Here it is required for activation of the checkpoint kinase Chk2, possibly through recruitment of the mediator protein 53BP1 to the site of damage [31]. Known post-translational modifications of the Cohesin subcomponents important for different aspects of DNA repair are schematically illustrated in Fig. 2B. Yeast Cohesin has not been reported to be involved in checkpoint signalling or maintenance. But in addition to its importance for DSB repair in G2 via HR, Cohesin has been suggested to regulate NHEJ in Saccharomyces cerevisiae. This was concluded from experiments on SMC1 mutated cells where NHEJ-based plasmid repair could not be performed correctly. This defect was rescued by deletion of the HR proteins Rad52 or Rad54, suggesting that Smc1 (and presumably Cohesin) regulate the choice between HR and NHEJ rather than directly taking part in the NHEJ process as such [32]. After concluding that S-phase cohesion is required for postreplicative DSB repair, it remained to be determined whether this was the sole repair function of Cohesin. In extensively laser-damaged human cell lines, fluorescently tagged Cohesin components could be detected in the track of the damage [33]. This type of damage causes an extremely high density of DSBs, putting the physiological relevance of this accumulation in question. It was however later shown that Cohesin is recruited to site-specific DSBs in yeast and human cells [34–36]. These observations indicated that the complex could be more actively involved in the repair process. If DNA-loading of Cohesin, and thereby localization to break, is inactivated in G2 after establishment of S-phase cohesion, no repair of DSB induced by γirradiation occurs [34,35]. This shows that even if S-phase established cohesion is fully functional, Cohesin also has to be present at the site of damage to allow repair. With an assay taking advantage of temperature sensitive S-phase cohesion and wild type Cohesin expressed in G2, it was also demonstrated that in response to DSBinduction after completion of replication, so-called damage-induced (DI-) cohesion was formed [34,37]. Is this cohesion required for postreplicative DSB repair? To facilitate the discussion of this matter, the details of cohesion establishment have to be described.

Cohesion establishment during S-phase To establish cohesion Cohesin first has to be loaded onto chromosomes. This occurs in G1 or early S-phase in yeast, and in telophase in mammalian cells [4,38–41]. The chromosome

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E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 1 4 45 – 1 45 3

Fig. 2 – An illustration of the cohesion machinery and its regulation through the cell cycle. (A) The Cohesin complex, where Smc1 and Smc3 monomers are folded at their hinge domains, and hinge and head domains are connected by the coiled coil regions. Smc1/ 3 dimers are formed via an interaction between their hinge domains and an ATP dependant connection at the heads. Scc1 further locks the Smc1/3 binding at the head domains and stably associates the Scc3 subunit to the complex. The cohesion regulatory proteins Pds5 and Wpl1 (Rad61) forms a complex with Scc3 where Wpl1 interacts directly with Scc3. (B) Factors required for loading of Cohesin, for establishment of cohesion and for its maintenance are framed. Known post-translational modifications of importance for induction of DNA damage checkpoints and for the state of cohesion are indicated. Information shown is based on experiments in yeast and HeLa cells. For further details see the text.

association in yeast depends on the ATPase activity of Smc1 and 3, as well as the Scc2/4 complex [12,42,43]. The Cohesin loading function of Scc2/4 appears to be conserved since in all species with

identified orthologs of Scc2/4, inhibition of their functions cause cohesion defects [8]. After loading, Cohesin is present on all centromeres and at specific sites along chromosome arms. In yeast,

E XP E RI ME N T AL C E L L R E S EA RC H 31 6 ( 20 1 0) 1 4 4 5– 1 45 3

arm Cohesin is mainly located in convergent intergenic regions with no or sparse sequence similarities [44,45]. In mammalian cells however, there is a substantial overlap between the GC rich consensus sites bound by Cohesin and CTCF, a zinc-finger protein required for transcriptional insulation [46–48]. Interaction between Cohesin and DNA is not sufficient for holding sister chromatids together. A number of proteins influencing establishment of cohesion have been identified, among which the acetyltransferase Eco1 seems to play the most central role [49–51]. Initially it was believed that cohesion could only be established during S-phase and there is indeed a strong connection with DNA replication [52,53]. The alternative RFC complex (Ctf8, Ctf18, Dcc1), the DNA polymerase associated protein Ctf4 and the DNA polymerase processivity factor PCNA (proliferating cell nuclear antigen) have all been implicated in cohesion establishment (for a review see [9]). Furthermore, Eco1 associates with the replication fork, and a physical interaction between the N-terminal part of Eco1 and PCNA is essential for cohesion establishment [54,55]. The acetyltransferase activity of Eco1 was first established in vitro where it was shown to acetylate itself and components of the Cohesin complex [51]. In vivo targets have however remained elusive, and only recently a number of important studies could show that the Eco1 mediated acetylation of two conserved lysines (K112, 113) in Smc3 is essential for cohesion establishment during S-phase (Fig. 2) [11,56–59]. Such modification of Smc3 seems to counteract an anti-establishment function performed by proteins previously shown to regulate cohesion maintenance, called Wpl1 (Rad61) and Pds5 [60–63]. This hypothesis was based on the fact that certain mutations in the WPL1 and PDS5 genes rescue the cohesion defect of ECO1 mutants. Interestingly, mutations in the Cohesin subunit SCC3 gene have also been shown to rescue such ECO1 mutants [11]. The anti-establishment and the cohesion maintenance function of Wpl1, Pds5 and Scc3 are proposed to act via the same mechanism, possibly inhibiting the opening of the Smc1–Smc3–Scc1 ring structure [11,56–59]. An early prediction for this type of involvement in cohesion establishment and maintenance was actually suggested for Pds5 in S. pombe, where PDS5 mutants rescued the defective cohesion phenotype of ESO1 mutants (ECO1 homolog in S. pombe) [64]. An additional protein of significance for cohesion regulation, present only in vertebrate cells, is called Sororin and is important for maintenance of the stably chromatin bound Cohesin [27].

Regulation of damage induced cohesion After replication is completed and S-phase cohesion has been established, Cohesin chromatin association and de-association continues. Thus, two fractions of Cohesin exist, one that is stably bound to DNA after replication and one that is constantly moving on and off DNA every 1 or 2 min [65,66]. However, in G2 newly loaded Cohesin becomes cohesive only if DNA is damaged. When a DSB is induced Cohesin is loaded specifically around the break. This recruitment depends on Scc2, as well as the DNA damage response factor Mre11, the kinases Tel1 and Mec1 and phosphorylation of the H2A histone (γ-H2A). These factors also influence the formation of damage-induced cohesion to different extents. As discussed, cohesion establishment during S-phase is strongly connected to DNA replication. Since Eco1 was shown to be required for DI-cohesion, and DSB repair via HR triggers DNA

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synthesis, it was a likely prediction that also this type of cohesion would depend on DNA synthesis for its formation. However, deletion of Rad52, required for strand invasion during HR (Fig. 1), did not influence the capability to generate cohesion in response to DSB [67,68]. Moreover, DI-cohesion turned out to be activated throughout the genome in response to a single DSB, further underscoring that cohesion can form independently of DNA synthesis, at least when triggered by DNA damage [67,68]. How and which signal is transmitted from a DSB to Cohesin to allow cohesion formation after replication? Koshland and coworkers found that the checkpoint kinase Chk1 is required for DIcohesion. The conserved serine residue (S83) of Scc1 was defined as a Chk1 target since the exchange of serine 83 to alanine (A), which cannot be phosphorylated, inhibited formation of DIcohesion. If S83 instead was exchanged to a phosphorylationmimicking aspartic acid (D), neither Chk1 nor DSB was required for induction of cohesion in G2. However, functional Eco1 was still needed as demonstrated by analysing the possibility of generating cohesion in the background of temperature sensitive eco1-203 at non-permissive temperature [69]. The phosphorylation of S83 by Chk1 was therefore suggested to augment Eco1-dependent acetylation of two critical residues in Scc1, K84 and K210. If both these sites are changed to arginine (R), which cannot be acetylated, DI-cohesion cannot be activated. If they are instead exchanged for glutamine (Q)(acetyl-mimic), cohesion can be generated in G2. This is true also in the absence of a DSB and with minimal acetyltransferase activity (eco1R222G, K223G(ack-) or eco1-203), implicating that acetylation of Scc1 is sufficient for formation of post-replicative cohesion. As for S-phase established cohesion, the acetylation of Cohesin seems to counteract the antiestablishment activity of Wpl1 [11,56–59,70]. Together this indicates that an intricate network of Cohesin modifications triggers the establishment of S-phase and DI-cohesion. However, not only does it remain to be shown that Scc1 phosphorylation and acetylation occur in vivo, the functional relevance of DI-cohesion is also somewhat unclear.

How important is DI-cohesion for DSB repair? To what extent does DI-cohesion contribute to DNA repair? If Eco1 is inactivated in G2, thereby preventing establishment of DIcohesion but leaving recruitment of Cohesin to break unaffected, DSB repair is abolished. This leads to the conclusion that recruitment of Cohesin to DSB is not sufficient, but that actual formation of DI-cohesion is required for this type of repair [67,68]. Accumulating data however suggest that this conclusion might be too simple. Several factors needed for full formation of DI-cohesion in G2 arrested cells have been shown to be dispensable for DSB repair in the same cell cycle phase, when analysed by pulse field gel electrophoresis. This assay is based on quantification of γ-ray induced breakage and subsequent recovery of whole yeast chromosomes [5]. A summary of the requirements for the recruitment of Cohesin to DNA breaks, for DI-cohesion and G2 DSB repair is presented in Table 1. Factors required for full formation of DI-cohesion, such as Tel1 and Mec1, are as shown in experiments done on cells lacking either of the proteins, not required for DNA repair (CS, LS unpublished data). However we find that a tel1 mec1 double mutant is severely repair-deficient indicating that these two kinases have overlapping roles in the

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Table 1 – Mutations and protein modifications of significance for DSB localization of Cohesin, formation of S-phase cohesion and/or DI-cohesion, and their importance for DNA repair. Mutation/modification

S-phase cohesion

Cohesin at DSB

DIcohesion

DNA repair

Comment

Ref

mre11Δ mec1Δ tel1Δ mec1Δ /tel1 Δ H2A S129stop rad53 Δ scc2-4 eco1-1 eco1-203 eco1 ack-(R222G, K223G) smc6-56 rad52 Δ rad9 Δ chk1 Δ

+ + + + + nt − − − + + + + +

− +/− +/− − − +/− + + + −, + ⁎ nt + +

− − +/− nt +/− nt − − − − − + + −

− + + − + Nt − − − − − − Nt +

35 35, † 35, †, 66,67 Tel1/Mec1 redundancy? 35, † 35, †, 66,67 Rad53 can P Scc1 35 34, 35, 66 66 Eco1wt overexpression induce G2 cohesion w/o DSB 67 In vivo acetylase activity remains 67 ⁎ - (human), +(yeast G2) 36, 66 66, 67 66 Chk1 P Scc1 in vitro 68

scc1-S83A (P-deficient)

+

+

−

−

scc1-S83D (P-mimic)

+

+

+

nt

P by Chk1 shown in vitro DI cohesion possible after overexpression of Eco1wt DI-cohesion in absence of DSB, also in bg of eco1ack-(R222G, K223G) but not in eco1-203 bg

scc1-K84R, K210R (Ac-deficient) scc1-K84Q, K210Q (Ac-mimic) rec8-N93S (creation of Chk1 P site) rec8-N93D (P-mimic) wpl1 Δ wpl1 Δ (scc1-K84R, K210R bg)

+ + + + +/− nt

+ + + (slow) ⁎ nt nt nt

− + + + + +

nt nt − nt nt nt

DI-cohesion in absence of DSB also in eco1-203 bg ⁎ localization of Rec8 DI-cohesion in absence of DSB DI-cohesion only after DSB induction

68 68 69 69 68 68 69 69

− deficient, + proficient, +/− moderate effect of deletion or mutation, nt = not tested, P = phosphorylation, Ac = acetylation, † CS, LS unpublished.

formation of DI-cohesion. Inhibition of H2A phosphorylation also impairs DI-cohesion but leaves DSB repair mostly unperturbed ([67], CS/LS unpublished). Similarly, Chk1 is important for formation of DI-cohesion but not essential for DSB repair in G2 ([69], CS/LS unpublished). Since deletions of TEL1 or MEC1 as well as preclusion of H2A phosphorylation to different extents diminish Cohesin accumulation at DSB [35], the lack of effect on DSB repair when deleting Tel1, Mec1 or preventing phosphorylation of H2A shows that full recruitment of Cohesin to the break is not absolutely required for repair. Finally, an acetylateable form of Rec8, the meiotic variant of Scc1, is localized to breaks, allows formation of DI-cohesion but still does not support DSB repair [69]. Together this reopens the question what Scc2 and Eco1 do to promote DSB repair. As stated, inactivation of these two in G2 arrested cells completely abrogates both DI-cohesion and DSB repair. One possibility is that they have other repair functions in addition to their roles in recruitment of Cohesin to breaks and/or DI-cohesion. It has since some time been known that the Scc2/ Cohesin system is important for gene regulation [71–73], and it is tempting to speculate over a possible function for DI-cohesion in regulation of the transcriptional response to DSB [74,75]. The Eco1 acetyltransferase activity could be needed not only for modification of Cohesin proteins but also of histones. Acetylation of histone tails in a region of DNA is known to create an open chromatin structure and influence the transcriptional activity in this region [76]. Furthermore acetylation of histones H3 and H4 have been shown to be important for DSB repair [77]. Finally, it is important to keep in mind that DI-cohesion has until now only been detected in haploid budding yeast cells. Whether this repair mechanism is conserved or specific for this organism

remains to be determined. In support of cohesion activation in response to damage in other organisms it was recently reported that a single enzymatic DSB increases the proximity of sister chromatids in the region close to the break in chicken DT40 cells [78]. Furthermore, alignment of sister chromatids is transiently enhanced in response to X-irradiation or Mitomycin C treatment in Arabidopsis thaliana [79]. In addition, inhibition of Sororin leads to inefficient DNA repair without affecting the level of chromatin bound Cohesin, implicating that it is the cohesion function that is required for repair also in mammalian cells [27]. Further work is however required to fully understand the role of Cohesin in DSB repair. Such investigations will not only increase the understanding of DSB repair in general, but might also shed light on the Cornelia de Lange and Roberts syndromes, caused by mutations in cohesionconnected genes [80,81].

Acknowledgments We would like to thank the members of our labs for continuous discussions on damage-induced cohesion and its possible importance. We would also like to apologize to colleges whose work we could not site correctly due to space limitations. The authors are supported by the Swedish Cancer Society, the Swedish Research council and Karolinska Institutet. L.S. is in addition funded by The Jeansson foundation. C.S. is a Royal Swedish Academy of Sciences Research Fellow supported by Knut and Alice Wallenberg Foundation, and is in addition supported by an ERC starting grant, Vinnova, and Swedish foundation for Strategic research.

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