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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
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?
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?
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).
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