Advances in understanding DNA processing and protection at stalled replication forks

Abstract

The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.

Figure 1.

Replication fork intermediates visualized by EM. To visualize replication fork intermediates, replicating cells are treated with the cross-linking agent psoralen, which cross-links DNA upon UVA exposure. The cross-linked duplex DNA is then visualized by EM, and replication intermediates are analyzed. ssDNA versus dsDNA is determined by measuring DNA fiber thickness. Progressing replication forks, reversed replication forks, and replication forks containing ssDNA gaps at the replication fork junction (thick black arrow) and behind the fork (thick white arrow) have been visualized. Scale bars indicate 0.5 kb in main images and 0.2 kb in insets. EM micrographs are reproduced with permission from Zellweger et al. (2015).

Figure 2.

Summary of proteins and their roles in processing or protecting stalled replication forks. Recent observations suggest that many proteins promote the remodeling of DNA at stalled replication forks into reversed replication fork structures. The remodeled fork requires protection by BRCA1, BRCA2, RAD51, and several other factors that have been identified. In the absence of replication fork protection, the newly synthesized DNA is subject to degradation by nucleases. A number of proteins have also been identified as promoting the localization of these nucleases at the stalled fork. Further investigation is required to determine how these fork remodelers, nuclease regulators, and fork protectors may be operating to promote replication fork stability. It is possible that the various remodelers, protectors, regulators, and nucleases are operating in a coordinated fashion; however, it is also possible that their roles are DNA lesion and replication fork structure dependent.

Figure 3.

Distinct roles of BRCA2 and RAD51 in canonical homologous recombination and replication fork protection. (A) HDR of DSBs requires the formation of 3′ ssDNA overhangs. The MRE11–RAD50–NBS1 (MRN) complex senses DSB and with the CtiP endonuclease initiates DNA end resection. The EXO1 exonuclease or the BLM–DNA2 helicase nuclease complex is responsible for more extensive resection. BRCA2 loads and stabilizes RAD51 nucleofilaments on the ssDNA overhangs displacing the ssDNA binding protein RPA. RAD51 nucleofilament invades the sister chromatid to perform homology search. DNA synthesis proceeds using homologous DNA for precise repair. (B) Replication fork reversal is proposed to be a global response to replication stress that requires RAD51 and BRCA2 for fork reversal and fork protection. RAD51-mediated fork reversal entails the annealing of the newly replicated (nascent) strands of DNA and reannealing of the parental DNA strands. This function is proposed to require RAD51 independently of BRCA2. Subsequently, both RAD51 and BRCA2 are necessary to prevent nascent strand degradation.

Figure 4.

RADX modulates RAD51 activity at replication forks. (A) A proposed model of RADX function is to regulate RAD51 activity at the replication fork to prevent unnecessary RAD51 association and fork remodeling during unperturbed DNA replication. Upon RADX depletion, there is increased genomic instability and DSBs that may be the result of inappropriate replication fork remodeling, leading to increased fork cleavage by MUS81. (B) The depletion of RADX in BRCA2-deficient cells rescues nascent strand degradation at HU stalled replication forks without affecting homologous recombination. It is proposed that the removal of RADX results in increased RAD51 function, supporting improved RAD51 fork protection and the prevention of nascent strand degradation by nucleases.

Figure 5.

Replication fork reversal mediated by translocases. Replication fork reversal is mediated in part by the SNF2 family chromatin remodelers SMARCAL1, ZRANB3, and HLTF. Whether the translocases work synergistically to reverse forks or operate on distinct fork structures is unclear and needs further investigation. HLTF is important for the polyubiquitination of PCNA that serves as a platform for recruitment of ZRANB3. SMARCAL1 is recruited to the replication fork through interaction with RPA.