Recovering a Stalled Replication Fork

Citation: Sabatinos, S. A. (2010) Recovering a Stalled Replication Fork. Nature Education 3(9):31

At a stalled DNA replication fork, how is replication restarted after DNA damage has been resolved?

Aa Aa Aa

A schematic diagram shows three pathways by which kinase proteins signal replication fork stalling. The pathway at left occurs in mammalian cells; the pathway in the middle occurs in S. cereviseae cells; and the pathway at right occurs in S. pombe cells. Each pathway is represented as a downward-pointing vertical arrow. Proteins are located in the same three positions along the arrows in each of the three pathways: the top position, the middle position, and the bottom position. The kinases in the top positions transmit a signal to the proteins in the middle positions, which transmit the signal to the kinases in the bottom position (the effector kinases). In mammals, the top kinase is ATR, the middle protein is claspin, and the effector kinase is CHK1. In S. cereviseae, the top kinase is MEC1, the middle protein is MRC1, and the effector kinase is RAD53. In S. Pombe, the top kinase is Rad3, the middle kinase is MRC1, and the effector kinase is CDS1.

Figure 1: Intra-S phase checkpoint signaling

When replication stress is encountered, as during HU exposure, signals are transmitted through a kinase cascade. The paths compared between species are shown, and the given proteins in the pathway have functional similarity between species. At the top, signals are transmitted through the apical kinase: ATR in vertebrates; Mec1 in Saccharomyces cerevisiae (budding yeast); Rad3 in Schizosaccharomyces pombe (fission yeast). These kinases form a complex with adaptor proteins such as Atrip (or Ddc2, or Rad26) and transmit signals through transducers Claspin (or Mrc1 in yeast). For the purpose of this review, the ultimate target is the effector kinase: CHK1 in vertebrates, Rad53 in budding yeast, and Cds1 in fission yeast.

© 2001 Nature Education Adapted from Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nature Cell Biology 3, 958–965 (2001) 10.1038/ncb1101-958. All rights reserved.

Scientists interested in DNA replication often study the dynamics of the replication fork (RF), a core structural component of DNA synthesis. In laboratory experiments focused on the cell cycle, hydroxyurea (HU) is used to model DNA damage during replication (S phase of the cell cycle). HU causes the depletion of deoxynucleotide triphosphates (dNTPs) and consequent RF stalling. The Y-shaped structure of the RF, which is at the center of action during DNA replication, coordinates unwinding and synthesizing of the template DNA. A host of proteins support the RF during this process and ensure that replication errors or DNA strand breakages are corrected before replication and the cell cycle proceeds further.

Some of the RF proteins are part of the fork protection complex (FPC), which also stabilizes the RF during HU-induced arrest. Single-stranded DNA (ssDNA) created at the stalled RF generates a signal that activates the intra-S phase checkpoint and prevents the cell cycle from moving into G₂ phase until replication is complete (Figure 1). Once the replication block has been removed, either by adaptation to the drug or by the removal of HU from the system, stalled RFs resume DNA synthesis.

Although some replication origins can start in late S phase (Lopes et al. 2001), completion of replication following arrest generally depends on restart of the stalled forks (Branzei & Foiani 2007). This reliance on restarting RFs to complete whole genome synthesis raises several important questions. What signal alerts the cell that replication has paused and RF progression has stopped? How do RFs restart replication once conditions improve or the error is resolved? These questions are particularly important to human health because maintenance of genome stability during S phase helps defend against cancer (Bartkova et al. 2005).

The Unexpected Pause: Signals from Fork Stalling

A simplified schematic diagram shows three examples of double-stranded DNA punctuated by single-stranded regions. Double-stranded DNA is represented as two thin, horizontal, blue lines arranged in parallel. Where the DNA is single-stranded, one of the horizontal blue lines is absent.

Figure 2: Single-stranded DNA (ssDNA) at the stalled replication fork

(A) RFs slow and then stall during HU treatment, generating ssDNA. (B) This ssDNA is a signal that resembles DNA damage from resected DNA DSBs or single-strand repair patches. (C) ssDNA is also generated at telomeres and is a signal of telomere instability. A common feature in all of these structures is the presence of ssDNA, which causes ATR activation to promote RF stalling, DNA repair, and cell cycle stalling. Thus, ssDNA is a potent signal that links DNA damage to repair.

© 2008 Nature Publishing Group Cimprich, K. A. & Cortez, D. ATR: an essential regulator of genome integrity. Nature Reviews Molecular Cell Biology 9, 616-627 (2008) doi:10.1038/nrm2450. All rights reserved.

RF stalling causes physical changes that induce checkpoint signals. For instance, even when RFs stall, the minichromosome maintenance (MCM) helicase continues unwinding the DNA and generates some excess ssDNA (Smith et al. 2009; Van et al. 2010). Replication protein A (Rpa) is an ssDNA-binding protein that keeps the DNA from reannealing and is recruited to coat ssDNA at the paused fork (Alcasabas et al. 2001; Kanoh et al. 2006; MacDougall et al. 2007; Van et al. 2010). In some ways, Rpa-coated ssDNA may signal DNA damage, because it resembles other types of DNA problems, such as resected ends of a DNA double-strand break, or DSB (Figure 2). Rpa-coated ssDNA also allows the Rad9/Rad1/Hus1 (9-1-1) complex to load (Kanoh et al. 2006; Zou et al. 2003). This complex looks and acts similarly to the replication factor PCNA (proliferating cell nuclear antigen) but is specific for damage response. Thus the Rpa-coated ssDNA brings together signaling components for downstream checkpoint response (Branzei & Foiani 2006; Kanoh et al. 2006; Kemp & Sancar 2009; Lucca et al. 2004; Yan & Michael 2009a, 2009b; Zou et al. 2003).

Some studies in budding yeast (Saccharomyces cerevisiae) have helped us understand the function of yet another protein that participates in RF stalling: the recombination protein Rad52. This protein is recruited to the RF following HU exposure (Lisby et al. 2001). Rad52 is a homologous recombination protein required to repair DSBs, and its recruitment during HU treatment confirms two features. One is that HU exposure causes DNA damage; the other is that restarting the stalled RF involves DNA recombination. During prolonged HU exposure, the recombination and repair protein Mre11 is also recruited, again reinforcing the contention that long-term RF arrest has deleterious effects on the DNA (Lisby et al. 2004).

However, RF stalling is not a permanent event. In fission yeast (Schizosaccharyomyces pombe) the Rpa signal at a stalled RF is lost over time, while the Rad52 homolog Rad22 is recruited (Irmisch et al. 2009). Rad52/Rad22 and Rad51 recombination proteins are known to displace Rpa during homologous recombination (Kurokawa et al. 2008; Seong et al. 2009; Sugiyama & Kantake 2009). So it appears that the ssDNA-Rpa signal times out and switches to ssDNA-Rad51 in order to promote recombination-dependent RF restart mechanisms. Is the HU-induced DNA damage reversible or permanent? At the level of single molecules, it appears the answer is that DNA damage persists. Single-molecule "DNA fiber" analysis demonstrated that DNA damage signals increase with extended HU treatment, and the damage signal persists even after HU is removed from the system (Petermann et al. 2010). It is clear that the replication checkpoint is linked to the DNA damage checkpoint, and DNA damage triggers the checkpoint signal that prohibits progression to metaphase (Figure 3). Perhaps the accumulation of damage and the inability to restart with prolonged HU exposure are due to the loss of components from stalled forks. Could a switch from Rpa to Rad51 be a signal to start repair and consequently restart the stalled RF?

A schematic diagram shows three pathways by which replication stress influences cell cycle checkpoints. The pathway at left occurs in S. cereviseae cells; the pathway in the middle occurs in S. pombe cells; and the pathway at right occurs in X. laevis cells. Each pathway is represented as a diagram of interconnected, downward-pointing vertical, horizontal, and diagonal arrows. Proteins are located in various positions between the arrows in each of the three pathways. In each pathway, replication stress is shown at the top of the diagram, and the cell cycle checkpoints affected by the signal cascade are shown at the bottom of the diagram.

Figure 3: Replication stress can affect two cell cycle checkpoints.

Alcabasas et al. (2001) demonstrated that the budding yeast protein Mrc1 is the replication checkpoint transducer, bringing Rad53 to Mec1. This action allows for Rad53 phosphorylation (activation), and triggers the S phase checkpoint. The process is similar in fission yeast: Mrc1 is the transducer of the phosphorylation signal from Rad3 to Cds1. In addition, Xenopus CLASPIN transduces the ATR signal to CHK1. However, prolonged RF stalling leads to DNA damage; alternatively, the fork may collapse if Rad53 (Cds1 in fission yeast, or Chk1 in metazoa) is absent, and such collapses cause DNA DSBs that cannot be repaired with RF restart. This DNA damage shifts the signal cascade to the DNA damage response branch. In yeast, the result is the activation of Chk1 proteins (which have a different role from metazoan Chk1) in a DNA damage-specific signaling pathway. Induction of the DNA damage pathway halts progression into metaphase while promoting DNA repair through appropriate repair mechanisms.

© 2001 Nature Publishing Group Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nature Cell Biology 3, 958-965 (2001) doi:10.1038/ncb1101-958. All rights reserved.

Recovery and Restart of DNA Replication

Once the block to replication is resolved, what signals the restarting of replication? One possible mechanism is protein phosphorylation, since several RF components are phosphorylated upon stalling (Figure 4). Mrc1, which is part of the FPC, is phosphorylated during fork stalling and then interacts with downstream effector kinases to transmit the checkpoint signal (see Figure 1; Bjergbaek et al. 2005; Lou et al. 2008; Xu et al. 2006). These protein modifications not only activate checkpoint response but can also attenuate the checkpoint, and Mrc1 is once again a good example. The Xenopus homolog of Mrc1, Claspin, is also phosphorylated by polo-like kinase, Plx1, causing Claspin to fall off of chromatin and stop the checkpoint signal (Yoo et al. 2004). In S. cerevisiae, Mrc1 phosphorylation changes its association with N- and C-terminal domains of the polymerase, Polε, which may modify polymerase association with the RF and its function (Lou et al. 2008). Are phosphorylated RF components simply modified, or are they lost and replaced at the fork? Does RF protein dephosphorylation reactivate the RF?

A schematic diagram shows an assemblage of proteins on a DNA replication fork. The proteins are depicted as shaded circles and ovals aggregated at the branching point on the DNA molecule, and along the leading, lagging strands. Proteins on the double-stranded region of the DNA molecule, to the right of the replication fork tines, are shown in a magnified inset below the first diagram. Beside them is the text: inhibition of origin firing.

Figure 4: ATR phosphorylation targets during replication fork stalling

When the RF stalls, the initial ssDNA signal activates ATR. Once activated, ATR kinase phosphorylates a variety of proteins to induce the replication checkpoint and promote RF stability. This diagram from Cimprich & Cortez (2008) illustrates the many targets of ATR phosphorylation. One such target is Chk1, the effector kinase responsible for activating the checkpoint and stalling the cell cycle, as seen in the simplified pathway of Figure 1. ATR is capable of phosphorylating many other substrates and potentially modifying their function, including RF components: MCM proteins, Rpa, polymerase, PCNA, and Claspin (Mrc1). These modifications alter protein association and function at the RF during stalling, and they are prime candidates for modifications that must be removed before replication resumes.

$A schematic diagram shows a replication fork in four stages of destabilization. The replication fork is depicted as two concentric circles: the outer circle is black, and the inner circle is grey. A replication fork at the top of the diagram is labeled \"bubbles.\" Below it, a second, more elongated replication fork is labeled \"large Ys.\" Below it, a more elongated replication fork is labeled \"residual DNA synthesis.\" Below it, a fourth, equally elongated replication fork is labeled \"replisome disassembly.\" In all of the replication forks, excluding the bottommost fork labeled \"replisome disassembly,\" a black circle is at the left and right side of each set of concentric circles. A shaded box is shown below the bottommost replication fork. In the top left of the box, a DNA structure is represented as two concentric circles. On the left side of the structure, the innermost concentric circle is bent and protruding into the hollow space inside the circles.$

Figure 5

The recombinase proteins Rad51 and Rad52 move to the nucleus with prolonged HU exposure and during restart, again emphasizing the essential role of recombination for restarting the fork (Hanada et al. 2007; Irmisch et al. 2009; Petermann et al. 2010; Torres et al. 2004; Wray et al. 2008). Corroborating evidence for this observation is that RAD51 mutant yeast cells (∆rhp51 in S. pombe) show reduced RF recovery and mitotic defects (Bailis et al. 2008). However, too much unchecked recombination can prevent fork restart (Bernstein et al. 2009; Liberi et al. 2005; Shimura et al. 2008; Willis & Rhind 2009; Yodh et al. 2009). Is there an intrinsic feature of the stalled RF that requires recombination activity to allow restart?

Checkpoint resolution and fork recovery depend on the creation of some ssDNA, as well as DNA damage repair, to restart replication. In addition, DNA nucleases help resolve stalled structures and are regulated by S phase checkpoint effector kinases: Chk1 in metazoa; Rad53 in Saccharomyces cerevisiae; Cds1 in Schizosaccharomyces pombe (Figure 1). Loss of these kinases results in excessive DNA unwinding in the presence of HU, producing an extended amount of ssDNA that becomes coated with RPA (Feng et al. 2006; Lucca et al. 2004; MacDougall et al. 2007; Sogo et al. 2002) and likely reflects uncoupling of the MCM helicase from the RF. Furthermore, the absence of effector kinases causes DNA damage with extensive formation of DSBs, which are likely caused by unresolvable DNA structures (Figure 5; Bailis et al, 2008; Feng et al. 2006; Lindsay et al. 1998; Lucca et al. 2004; Marchetti et al. 2002; Sogo et al. 2002).

Summary

The FPC stabilizes the RF during stalling. Protein modifications (e.g., phosphorylation) set up a signal cascade to promote stalling and maintain the fork structure. RF stalling must occur regularly during genome duplication, even in the absence of external DNA-damaging agents, resulting simply from sequence repetition or collision with DNA metabolism enzymes (Baird 2008; Lee et al. 2007; Mirkin & Mirkin 2007; Samadashwily et al. 1997). The stalling signal is deactivated when cells enter conditions allowing fork restart after DNA damage has been resolved. In addition, recombination and repair proteins are involved in replication restart, creating and repairing DNA breaks so that the fork can resume synthesis later. Research in yeast and metazoan systems to understand the nature of the stalled fork and restart due to HU is advancing our understanding of the processes involved, as well as the problems that develop when components are missing. The interplay between checkpoint signaling and break induction/recombination regulates HU response and stability in treated cells. Above, we discussed several detailed mechanisms for managing the quality of DNA replication, and the implications of these findings have a major impact on our understanding of how cells prevent carcinogenesis (Bartkova et al. 2005).

References and Recommended Reading

Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nature Cell Biology 3, 958–965 (2001)

Bailis, J. M. et al. Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. Molecular and Cellular Biology 28, 1724–1738 (2008)

Baird, D. M. Telomere dynamics in human cells. Biochimie 90, 116–121 (2008)

Bartkova, J. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005)

Bernstein, K. A. et al. Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair. EMBO Journal 28, 915–925 (2009)

Bjergbaek, L. et al. Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. EMBO Journal 24, 405–417 (2005)

Branzei, D. & Foiani, M. Interplay of replication checkpoints and repair proteins at stalled replication forks. DNA Repair (Amsterdam) 6, 994–1003 (2007)

Branzei, D. & Foiani, M. The Rad53 signal transduction pathway: Replication fork stabilization, DNA repair, and adaptation. Experimental Cell Research 312, 2654–2659 (2006)

Cimprich, K. A. & Cortez, D. ATR: An essential regulator of genome integrity. Nature Reviews Molecular and Cellular Biology 9, 616–627 (2008)

Feng, W. et al. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nature Cell Biology 8, 148–155 (2006)

Froget, B. et al. Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint. Molecular Biology of the Cell 19, 445–456 (2008)

Hanada, K. et al. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nature Structural & Molecular Biology 14, 1096–1104 (2007)

Hiyama, T. et al. Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells. Nucleic Acids Research 34, 880–892 (2006)

Irmisch, A. et al. Smc5/6 maintains stalled replication forks in a recombination-competent conformation. EMBO Journal 28, 144–155 (2009)

Kai, M. et al. Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. Genes & Development 19, 919–932 (2005)

Kanoh, Y., Tamai, K. & Shirahige, K. Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks. Gene 377, 88–95 (2006)

Kemp, M. & Sancar, A. DNA distress: Just ring 9-1-1. Current Biology 19, R733–R734 (2009)

Kurokawa, Y. et al. Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators. PLoS Biology 6, e88 (2008)

Lee, J. A., Carvalho, C. M. & Lupski, J. R. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 131, 1235–1247 (2007)

Liberi, G. et al. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes & Development 19, 339–350 (2005)

Lindsay, H. D. et al. S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes & Development 12, 382–395 (1998)

Lisby, M., Rothstein, R. & Mortensen, U. H. Rad52 forms DNA repair and recombination centers during S phase. PNAS 98, 8276–8282 (2001)

Lisby, M. et al. Choreography of the DNA damage response: Spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004)

Lopes, M. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001)

Lou, H. et al. Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Molecular Cell 32, 106–117 (2008)

Lucca, C. et al. Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing. Oncogene 23, 1206–1213 (2004)

MacDougall, C. A. et al. The structural determinants of checkpoint activation. Genes & Development 21, 898–903 (2007)

Marchetti, M. A. et al. A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway. PNAS 99, 7472–7477 (2002)

Mirkin, E. V. & Mirkin, S. M. Replication fork stalling at natural impediments. Microbiology and Molecular Biology Reviews 71, 13–35 (2007)

Petermann, E. et al. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Molecular Cell 37, 492–502 (2010)

Samadashwily, G. M., Raca, G. & Mirkin, S. M. Trinucleotide repeats affect DNA replication in vivo. Nature Genetics 17, 298–304 (1997)

Seong, C. et al. Regulation of Rad51 recombinase presynaptic filament assembly via interactions with the Rad52 mediator and the Srs2 anti-recombinase. Journal of Biological Chemistry 284, 24363–24371 (2009)

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. Journal of Molecular Biology 375, 1152–1164 (2008)

Smith, K. D., Fu, M. A. & Brown, E. J. Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis. Journal of Cell Biology 187, 15–23 (2009)

Sogo, J. M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002)

Sugiyama, T. & Kantake, N. Dynamic regulatory interactions of rad51, rad52, and replication protein-a in recombination intermediates. Journal of Molecular Biology 390, 45–55 (2009)

Torres, J. Z., Schnakenberg, S. L. & Zakian, V. A. Saccharomyces cerevisiae Rrm3p DNA helicase promotes genome integrity by preventing replication fork stalling: Viability of rrm3 cells requires the intra-S-phase checkpoint and fork restart activities. Molecular and Cellular Biology 24, 3198–3212 (2004)

Van, C. et al. Continued primer synthesis at stalled replication forks contributes to checkpoint activation. Journal of Cell Biology 189, 233–246 (2010)

Willis, N. & Rhind, N. Mus81, Rhp51(Rad51), and Rqh1 form an epistatic pathway required for the S-phase DNA damage checkpoint. Molecular Biology of the Cell 20, 819–833 (2009)

Wray, J. et al. Distinct RAD51 associations with RAD52 and BCCIP in response to DNA damage and replication stress. Cancer Research 68, 2699–2707 (2008)

Xu, Y. J., Davenport, M. & Kelly, T. J. Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast. Genes & Development 20, 990–1003 (2006)

Yan, S. & Michael, W. M. TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks. Journal of Cell Biology 184, 793–804 (2009a)

Yan, S. & Michael, W. M. TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation. Cell Cycle 8, 2877–2884 (2009b)

Yodh, J. G. et al. BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation. EMBO Journal 28, 405–416 (2009)

Yoo, H. Y. et al. Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase. Cell 117, 575–588 (2004)

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. Molecular and Cellular Biology 25, 563–574 (2005)

Zou, L., Liu, D. & Elledge, S. J. Replication protein A-mediated recruitment and activation of Rad17 complexes. PNAS 100, 13827–13832 (2003)