Arrested replication fork processing: Interplay between checkpoints and recombination
Introduction
The maintenance of genomic stability is a fundamental aspect of all life. DNA is damaged regularly by both external factors such as radiation and internal events such as oxidation. All cells devote a significant percentage of their genome to encoding DNA repair and DNA damage tolerance proteins that act in multiple complex and interrelated pathways to minimise the deleterious effects of such DNA damage. A cell is particularly vulnerable when it is replicating the DNA. DNA damage can block the passage of the polymerase, as can other events such as tightly bound non-histone proteins and clashes between the replication and transcriptional apparatus.
In addition to overcoming unexpected blocks, DNA replication must be tightly regulated to ensure that each base is only replicated once per cell cycle and to ensure that when replication forks encounter problems this does not result in a region of unreplicated DNA. In eukaryotic cells DNA replication is initiated at multiple origins and thus a single arrested fork will not necessarily prevent completion of replication because a fork initiated from an adjacent origin will replicate up to the site of arrest. However, if two converging forks experience arrest there is potential for an unreplicated region. Cells have developed many ways of overcoming this problem including the existence of late firing and cryptic origins of replication. Usually, such origins do not fire and are dismantled by passive replication from an adjacent efficient origin. When this dismantling does not occur, such origins have the capacity to fire and ensure the completion of replication. None the less, it is easy to imagine that there will be some circumstances replication cannot be completed.
To minimise this possibility, cells have established mechanisms to stabilise an arrested fork in such a way as to protect the newly synthesised DNA ends and to retain all the enzymatic machinery at the site of DNA incorporation. This ensures that the fork can restart when the problem is resolved (in this review we define these as a “stalled forks”). Where fork stalling does not occur correctly, or in circumstances where the replication problem is directly associated with loss of the replication proteins (defined here as “collapsed forks”), cells resort to a number of DNA processing events that may act to protect the fork from further inappropriate DNA processing and/or to restore replication. In this review we will focus on the checkpoint pathways that act to stabilise arrested forks and the recombination pathways that can process forks that collapse.
Section snippets
Error-free replication restart in prokaryotic cells
Error-free replication restart processes have been extensively studied in E. coli and the existence of a recombination-dependent pathway has been clearly established [1]. Recombination permits cells to reassemble a functional replisome at a collapsed fork and to re-establish unidirectional replication independently of the origin of replication [2], [3], [4]. This can be summarised as follows: recombination proteins act at stalled forks to generate recombination intermediates (such as a D-loop)
Fork stabilization by the DNA replication checkpoint in yeast
The DNA replication checkpoint is essential to maintain genome integrity under stress conditions and during unchallenged replication. The replication checkpoint ensures complete chromosome replication by a combination of effects; entry into mitosis is delayed to allow time for replication to be completed. Firing of late replication origins is prevented to stop the formation of new replication forks. Active replication forks are stabilised to ensure replication can progress once the stress is
Stalled fork processing by recombination proteins in yeast
Several lines of evidence suggest that recombination is important for recovery from replication perturbation. Genetic interaction studies showed that the viability of several replication and replication checkpoint mutants depends on recombination process, suggesting that recombination can help to resume replication [63], [64], [65], [66], [67], [68]. Recombination mutants also show reduced viability when exposed to agents that compromise replication such as HU, UV or MMS. Analysis of
Co-ordination of replication and recombination by checkpoint proteins
Recombination protects fork integrity in bacteria. In eukaryotes, preservation of fork integrity is largely dependent on checkpoint proteins. In E. coli, replication forks break in absence of recombination. In eukaryotes breakage occurs in absence of checkpoint or in the absence of RecQ family helicases [109], [159]. The observation that, in eukaryotes, HU-arrested forks experience unscheduled Rhp51Rad51-dependent recombination in absence of the checkpoint suggests that stalled forks are first
Conclusion
In conclusion, how arrested replication forks are processes by checkpoint and recombination pathways has been extensively studied in both S. cerevisiae and S. pombe. Despite this, these mechanisms are not well understood. It is clear that the nature of the RFB influences how replication is resumed (Fig. 5). When replication forks are stalled by the replication inhibitor hydroxyurea, checkpoints proteins are activated, leading to replisome stabilization. If this checkpoint fails and the
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