Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Institute of Cancer Biology and Center for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
The Journal of Cell Biology (Impact Factor: 9.83). 10/2009; 186(5):655-63. DOI: 10.1083/jcb.200812138
Source: PubMed


Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA-mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.

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Available from: Jacob Falck Hansen, Apr 16, 2014
    • "It is unclear why depletion of two independent factors (namely BLM or FBH1) is able to compensate for a lack of BOD1L, although BLM and FBH1 have partially redundant functions in DT40 cells (Kohzaki et al., 2007). While FBH1 and BLM both have pro-and anti-recombinogenic activities (Bugreev et al., 2007; Fugger et al., 2009), BLM can displace RAD51 from ssDNA and can also potentiate HR through its ability to stimulate DNA2-dependent end-resection by binding to RPA (Chen et al., 2013; Xue et al., 2013; Sturzenegger et al., 2014). It is possible that loss of BLM activity in BOD1L/BLM knockdown cells has two effects: (1) increases RAD51 filament stability and; (2) compromises DNA2- dependent resection of damaged forks, the latter of which causes the genome instability apparent in BOD1L-deficient cells. "
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    ABSTRACT: Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 07/2015; 59(3). DOI:10.1016/j.molcel.2015.06.007 · 14.02 Impact Factor
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    • "Cells were grown on coverslips and treated as indicated in the figure legends. The coverslips were fixed and stained as described previously (Fugger et al., 2009). Briefly, the coverslips were fixed in Lillies with or without prior preextraction in CSK buffer (0.5% Triton X-100 in 20 mM HEPES [pH 7.4], 50 mM NaCl, 3 mM MgCl, and 300 mM sucrose), permeabilized in 0.2% Triton X-100/PBS, and incubated with the indicated antibodies diluted in DMEM supplemented with 10% serum. "
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    ABSTRACT: DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork in vitro and promotes fork regression in vivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 03/2015; 10(10). DOI:10.1016/j.celrep.2015.02.028 · 8.36 Impact Factor
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    • "The closest sequence homolog of Srs2 in mammals and other vertebrates is FBH1, which is also found in fission yeast but not in budding yeast. Several lines of in vivo evidence suggest that this UvrD-type helicase regulates HR at the stage of RAD51 filament assembly, but its role in SDSA is yet to be assessed (18). Another potential ortholog of Srs2 in mammals is RECQ5, which belongs to RecQ family of DNA helicases (19). "
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    ABSTRACT: Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.
    Nucleic Acids Research 12/2013; 42(4). DOI:10.1093/nar/gkt1263 · 9.11 Impact Factor
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