Accurate and complete genome duplication is crucial to maintain cell survival and prevent malignant transformation. The Fanconi anemia (FA) pathway has traditionally been associated with the repair of DNA interstrand crosslinks that impede the progression of the replication machinery. Recent studies demonstrate that FA proteins also regulate cell-cycle checkpoints and/or promote replication fork remodeling in response to multiple DNA impediments, and redefine the FA pathway as a fundamental mechanism to preserve genome integrity upon different insults. Alterations in FA genes fuel genomic fragility and constitute a driving force of tumorigenesis. We highlight current understanding of FA signaling in safeguarding genome stability during replication, and discuss the identification of novel determinants of cancer cell survival in FA-deficient tumors. 

Fanconi anemia proteins and genome fragility: unraveling replication defects for cancer therapy.

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication. Description Editor’s summary DNA replication is challenged by obstacles, including DNA damage, that impede synthesis and threaten genome stability. A common response to this replication stress is replication fork reversal, in which parental DNA reanneals and a new daughter strand duplex is formed. Liu et al. describe a mechanism dependent on the recombinase RAD51 by which cells can accomplish reversal without unloading the replication machinery, including the helicase complex, which cannot be reloaded once replication has started. This mechanism explains how reversal helps cells tolerate replication stress and rapidly resume DNA synthesis once the replication obstacle is removed. —Di Jiang Fork reversal requires the RAD51 recombinase to overcome the CMG helicase, which remains bound to the stalled fork.

RAD51 bypasses the CMG helicase to promote replication fork reversal

Summary FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

https://biorxiv.org/content/10.1101/2021.07.08.450798v1.full.pdf

Carl A. Sedgeman

Papers

FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange