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Recovering a Stalled Replication Fork

By: Sarah A. Sabatinos, Ph.D. (Dept. of Molecular and Computational Biology, University of Southern California) © 2010 Nature Education 
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?
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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. View Terms of Use
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. View Terms of Use

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.
© 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. View Terms of Use

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).


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

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