Preventing replication fork collapse to maintain genome integrity

Abstract

Billions of base pairs of DNA must be replicated trillions of times in a human lifetime. Complete and accurate replication once and only once per cell division cycle is essential to maintain genome integrity and prevent disease. Impediments to replication fork progression including difficult to replicate DNA sequences, conflicts with transcription, and DNA damage further add to the genome maintenance challenge. These obstacles frequently cause fork stalling, but only rarely cause a failure to complete replication. Robust mechanisms ensure that stalled forks remain stable and capable of either resuming DNA synthesis or being rescued by converging forks. However, when failures do happen the fork collapses leading to genome rearrangements, cell death and disease. Despite intense interest, the mechanisms to repair damaged replication forks, stabilize them, and ensure successful replication remain only partly understood. Different models of fork collapse have been proposed with varying descriptions of what happens to the DNA and replisome. Here, I will define fork collapse and describe what is known about how the replication checkpoint prevents it to maintain genome stability.

Keywords: ATR; Fork collapse; Replication checkpoint; Replisome; iPOND.

Figure 1

Three models of how the replication checkpoint prevents fork collapse. See text for details.

Figure 2

Fork stalling causes a decrease in PCNA levels associated with replication forks. After an Okazaki fragment is completed and chromatin is deposited, PCNA is unloaded from the lagging strand. When a fork is elongating normally, PCNA is rapidly placed back onto the lagging strand to support generation of a new Okazaki fragment and continued DNA synthesis. When the fork stalls due to nucleotide depletion, PCNA is no longer loaded since new Okazaki fragments are not being initiated rapidly. The PCNA that was already on the lagging strand is unloaded so the total amount of PCNA at the fork is less. This model predicts that the amount of PCNA at forks DNA should be higher in normal replicating cells than in HU-treated cells. This is exactly what is observed when the amount of PCNA associated with the nascent DNA is examined by iPOND [56].

Figure 3

The checkpoint regulates fork regression and enzymatic cleavage at stalled replication forks. Checkpoint-deficient cells have excessive or persistent fork regression creating substrates for structure-specific nucleases. Nuclease action generates double-strand breaks and excessive ssDNA when the checkpoint is inactive.