The DNA damage response during DNA replication

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Eukaryotic chromosome replication is mediated by multiple replicons and is coordinated with sister chromatid cohesion, DNA recombination, transcription and cell cycle progression. Replication forks stall or collapse at DNA lesions or problematic genomic regions, and these events have often been associated with recombination and chromosomal rearrangements. Stalled forks generate single-stranded DNA that activates the replication checkpoint, which in turn functions to protect the stability of the fork until the replication can resume. Recombination-mediated and damage-bypass processes are the main mechanisms responsible for replication restart. New findings have helped to unmask the molecular mechanisms that sense replication stress, control the stability of replication forks, and regulate the mechanisms that promote replication restart, thereby giving us a better understanding of how genome integrity is preserved during replication.

Introduction

Most of the chromosomal abnormalities arising in cancer cells are caused by faulty chromosome replication [1]. DNA replication represents a dangerous moment in the life of the cell as endogenous and exogenous events challenge genome integrity by interfering with the progression, stability and restart of the replication fork. To deal with this responsibility, replication forks are endowed with an extraordinary potential to coordinate fork stalling with fork resumption processes. Failure to protect stalled forks or to process the replication fork appropriately for replication restart results in the accumulation of mutations and genomic aberrations. Indeed, a variety of human genetic syndromes that lead to cancer predisposition are caused by mutations in genes that protect the genome integrity during chromosome replication.

In this review we will comment on the recent findings that helped to elucidate how stalled forks signal to the replication checkpoint, how the checkpoint mechanisms contribute to the stability of the fork, the mechanisms that assist and coordinate fork restart, and the enzymatic activities that process stalled or collapsed forks.

Section snippets

Endogenous and exogenous events causing replication fork stalling and collapse

Replication fork progression is slowed down at several genomic sites, such as tRNA genes [2], specialized protein-mediated replication fork barriers [3, 4], replication slow zones [5] and inverted repeats [6••]. These chromosomal loci are known as fragile sites and induce fork pausing, which is often associated with chromosome breakage and genomic rearrangements [5]. Fork pausing can also be caused by intra-S DNA damage through several mechanisms: by causing uncoupling between the replisome and

Sensing replication stress: signals, thresholds and limitations of the checkpoint mechanism

Stalled forks promote checkpoint activation by exposing significant amounts of single-stranded DNA (ssDNA) coated by replication protein A (RPA) [15, 16, 17, 18••]. It is becoming clear that it is not the damaged DNA per se that generates the checkpoint signal but rather the collision of the fork with the lesion (Figure 1). Recent observations show that functional uncoupling of the MCM helicase and polymerase activities at the fork is required for generation of RPA–ssDNA and checkpoint

Checkpoint-mediated stabilization of stalled forks

One of the most important and so far best-studied mechanisms guarding genomic integrity during S phase is provided by replication checkpoints. Electron microscopic analysis of the budding yeast rad53 mutant showed that an important function of the replication checkpoint is to protect the stability of stalled forks [15]. In checkpoint mutants, stalled forks rapidly degenerate, accumulating gapped and hemireplicated molecules [15]. These long ssDNA regions seem to result from lagging strand

Fork restart mechanisms

Unlike E. coli, which rely on a single replication origin and need to engage the collapsed forks into replication-coupled recombination processes in order to complete replication, eukaryotic organisms are endowed with several routes to restart the fork (Figure 3).

Recombination-mediated replication-restart and damage-bypass mechanisms are frequently used by eukaryotic organisms to replicate damaged DNA or to resume replication after fork collapse. Recombination mechanisms assist completion of

Conclusions

The past few years have brought significant contributions in understanding the molecular basis of replication initiation, how replication problems are sensed and how the replication forks are appropriately stabilized or restored in order to prevent genomic aberrations. Important issues, however, such as mitochondrial replication and the coordination between replication progression and recombination induction in the meiotic cycle, still await elucidation. Recent advances in imaging, microarray

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The authors apologize for the many interesting articles that they were not able to discuss or acknowledge due to space limitations. We thank all members of Foiani's laboratory and Joel A. Huberman for useful discussions, and Katsuhiko Shirahige for communicating unpublished results. The work carried out in Foiani's laboratory was supported by grants of the Italian Association for Cancer Research and the European Community, and D. Branzei was supported by the RIKEN Special Postdoctoral Research

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