Studying Homologous Recombination in Fanconi Anemia

ABSTRACT The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased ∼9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

Download Full-text

Loss of the homologous recombination generad51leads to Fanconi anemia-like symptoms in zebrafish

10.1101/095646 ◽

2016 ◽

Author(s):

Jan Gregor Botthof ◽

Ewa Bielczyk-Maczyńska ◽

Lauren Ferreira ◽

Ana Cvejic

Keyword(s):

Bone Marrow ◽

Homologous Recombination ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Tumor Development ◽

Embryonic Stem ◽

Dominant Negative ◽

Hematopoietic Stem ◽

Recombination Protein

AbstractRAD51is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterized by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. In this paper, we describe the first viable vertebrate model ofRAD51loss. Zebrafishrad51loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Co-mutation ofp53was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment ofRAD51as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.Significance statementThe homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in anin-vivocontext in vertebrates. Here we report the first viable vertebrate mutant model ofrad51in zebrafish. These mutant fish enabled us to confirm for the first time the recently discovered role ofRAD51in Fanconi anemia pathogenesis. We report that p53 linked embryonic stem cell defects directly lead to hematological impairments later in life. Co-mutation ofrad51withp53rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

Download Full-text

Fanconi anemia and homologous recombination gene variants are associated with functional DNA repair defects in vitro and poor outcome in patients with advanced head and neck squamous cell carcinoma

Oncotarget ◽

10.18632/oncotarget.24797 ◽

2018 ◽

Vol 9 (26) ◽

pp. 18198-18213 ◽

Cited By ~ 8

Author(s):

Caroline V.M. Verhagen ◽

David M. Vossen ◽

Kerstin Borgmann ◽

Floor Hageman ◽

Reidar Grénman ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Dna Repair ◽

Homologous Recombination ◽

Cell Carcinoma ◽

Fanconi Anemia ◽

Gene Variants ◽

Advanced Head ◽

Functional Dna ◽

Recombination Gene

Download Full-text

Faculty Opinions recommendation of Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023441.269754 ◽

2005 ◽

Author(s):

David Schatz

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Gene Conversion ◽

Fanconi Anemia ◽

Immunoglobulin Gene

Download Full-text

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector

The Journal of Gene Medicine ◽

10.1002/jgm.1382 ◽

2009 ◽

Vol 11 (11) ◽

pp. 1012-1019 ◽

Cited By ~ 6

Author(s):

Kittiphong Paiboonsukwong ◽

Fumi Ohbayashi ◽

Haruka Shiiba ◽

Emi Aizawa ◽

Takayuki Yamashita ◽

...

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Hematopoietic Cells ◽

Virus Vector ◽

Adeno Associated Virus ◽

Fanconi Anemia Gene

Download Full-text

Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽

10.1182/blood.v108.11.186.186 ◽

2006 ◽

Vol 108 (11) ◽

pp. 186-186

Author(s):

Natalie B. Collins ◽

Andrei Tomashevski ◽

Gary M. Kupfer

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Phosphorylation Site ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Damage Response ◽

And Function ◽

Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

Download Full-text