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Human Molecular Genetics Advance Access originally published online on August 31, 2005
Human Molecular Genetics 2005 14(20):3027-3033; doi:10.1093/hmg/ddi334
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fancd2 functions in a double strand break repair pathway that is distinct from non-homologous end joining

Scott Houghtaling1,*, Amy Newell1, Yassmine Akkari1, Toshiyasu Taniguchi2, Susan Olson1 and Markus Grompe1

1Department of Molecular and Medical Genetics L103, Oregon Health and Science University, Portland, OR 97239, USA and 2Division of Human Biology and Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA

* To whom correspondence should be addressed at: Department of Molecular and Medical Genetics L103, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Tel: +1 5034946889; Fax: +1 5034946886; Email: houghtal{at}ohsu.edu

Received June 21, 2005; Accepted August 26, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fanconi anemia (FA) is a multigenic recessive disease resulting in bone marrow failure and increased cancer susceptibility. Cells from FA patients and mouse models are sensitive to DNA interstrand crosslinks (ICLs) and FA mice are moderately sensitive to ionizing radiation (IR). Both kinds of damage induce DNA double strand breaks (DSBs). To date, nine genes in 11 complementation groups have been identified; however, the precise function of the FA pathway remains unclear. Many of the proteins form a nuclear complex necessary for the mono-ubiquitination of the downstream protein, Fancd2. To further investigate the role of the FA pathway in repair of DSBs, we generated Fancd2–/–/Prkdcsc/sc double mutant mice. Prkdcsc/sc mutant mice have a defect in non-homologous end joining (NHEJ) and are sensitive to IR-induced DNA damage. Double mutant animals and primary cells were more sensitive to IR than either single mutant, suggesting that Fancd2 operates in DSB repair pathway distinct from NHEJ. Fancd2–/–/Prkdcsc/sc double mutant cells were also more sensitive to DSBs generated by a restriction endonuclease. The role of Fancd2 in DSB repair may account for the moderate sensitivity of FA cells to irradiation and FA cells sensitivity to ICLs that are repaired via a DSB intermediate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fanconi anemia (FA) is a rare autosomal and X-linked recessive disease resulting in birth defects, bone marrow failure and cancer susceptibility (1Go). Cells from patients and FA mouse models share a common phenotype of chromosomal instability, particularly following treatment with agents that induce interstrand crosslinks (ICLs) (2Go). At least 11 complementation groups exist, FA-A, B, C, D1, D2, E, F, G, I, J and L (3Go), and nine causative genes have been identified, including FANCA, FANCB, FANCC, FANCD1/BRCA2, D2, E, F, G/XRCC9 and L/PHF9 (4Go–12Go). Many of the FANC proteins interact to form a multisubunit nuclear complex required for the mono-ubiquitination of FANCD2 at lysine 561 that occurs following DNA damage and during the S-phase of the cell cycle (13Go,14Go).

FANC proteins have been suggested to have a role in the cellular responses to double strand breaks (DSBs) on the basis of reports that FA mice have a modestly increased sensitivity to ionizing radiation (IR) (15Go–17Go) and plasmid-based studies, which demonstrate an impaired processing of broken DNA ends in FA cells (18Go–20Go). DSBs are particularly toxic DNA lesions for which multiple repair systems have evolved. Two major pathways exist in eukaryotic cells, homology directed repair (HDR) and non-homologous end joining (NHEJ) (21Go,22Go). HDR is the major pathway for repair of DSBs in yeast, whereas NHEJ is the preferred pathway in mammalian cells (23Go,24Go). HDR subclasses include single-strand annealing and short-tract gene conversion, both of which have specific protein requirements (25Go–28Go). NHEJ also requires a distinct set of proteins including the main components, XRCC4/DNA ligase IV and DNA-PK, comprised of the DNA-PK catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer (21Go). The requirement for DNA-PK in NHEJ is supported by the phenotype of severe combined immune deficient (scid) mice, which have reduced DNA-PK activity due to a nonsense mutation in Prkdc (29Go). The NHEJ defect in these mice contributes to impaired V-D-J immunoglobulin recombination and increased sensitivity to IR-induced DSBs (30Go).

The FA field has slowly uncovered clues that suggest a specific role of FANCD2 in repair of DSBs by a homology-dependent mechanism that is distinct from NHEJ. First, the end joining activity deficient in FA extracts is not blocked by wortmannin in normal extracts, indicating that it is biochemically distinct from NHEJ (19Go). Secondly, FANCD2 is directed to nuclear foci containing the central recombination protein, RAD51 (14Go). The functional significance of this finding remains unclear, as vertebrate cells lacking FANCD2 have normal levels of RAD51 foci formation (17Go,31Go–33Go). Thirdly, FANCD2 was shown to directly interact with FANCD1/BRCA2, which binds to and modulates the activity of RAD51 (34Go). Next, recent experiments using chromosomally integrated reporter constructs have demonstrated a role of both human and chicken FANCD2 in HDR of DSBs (32Go,33Go,35Go). Finally, FANCD2 has been shown to bind Holliday junctions and double strand DNA substrates in vitro (36Go).

In this study, we undertook a genetic approach to examine the epistatic relationship between Fancd2 and Prkdc in mammals. We show that Fancd2 operates in a DSB repair pathway that is distinct from Prkdc, thus Fancd2 and Prkdc are non-epistatic for DSB repair. We also show that NHEJ plays little to no role in the repair of ICLs. Finally, we show that Fancd2–/–/Prkdcsc/sc double mutant cells are more sensitive to DSBs generated by a restriction endonuclease than Fancd2 complemented control cells, demonstrating that Fancd2 functions specifically in the repair DSBs rather than other types of DNA damage generated by irradiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fancd2 operates in a DSB repair pathway that is distinct from Prkdc-dependent NHEJ
We have previously described a role of Fancd2 in repair of ICLs; however, fibroblasts from Fancd2 knockout mice were not differentially sensitive to IR (17Go). In addition, Fancd2–/– mice were only moderately more sensitive than littermate controls to whole body irradiation (17Go). To investigate the relationship between the FA pathway and NHEJ, we crossed Fancd2 mutant mice to Prkdcsc/sc (scid) mice. Prkdcsc/sc mice are radiosensitive because of the reduced activity of the catalytic subunit of DNA-PK, a central component of the NHEJ repair pathway (30Go,37Go). We predicted that if Fancd2 functions in a repair pathway that is separate from NHEJ, double mutant animals may be more sensitive to IR than Prkdcsc/sc mutants. To investigate this possibility, double mutant Fancd2–/–/Prkdcsc/sc mice and controls on a C57/Bl6J background were generated.

Primary ear fibroblasts prepared from Fancd2–/–/Prkdcsc/sc mice and controls were subjected to increasing doses of the 4'-hydroxymethyl-4,5',8-trimethylpsoralen plus UVA (HMT+UVA) in a cell growth assay (Fig. 1A). As expected for cells lacking an intact FA pathway, both Fancd2–/– and Fancd2–/–/Prkdcsc/sc mouse ear fibroblasts (MEFs) were highly sensitive to HMT+UVA-induced ICLs. Prkdcsc/sc cells were not more sensitive than wild-type controls and double mutant cells were not more sensitive than Fancd2–/– cells. Taken together, these results indicate that NHEJ plays little or no role in the repair of HMT+UVA-induced ICLs in primary mouse fibroblasts.



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  		Figure 1. DNA damage sensitivity of Fancd2–/–/Prkdcsc/sc cells and controls. Primary MEFs of the indicated genotypes were plated in quadruplicated and treated with increasing doses of HMT+UVA (A) or IR (B). Total DNA content was measured as an indication of cell growth. Double mutant cells were more sensitive to IR than Prkdcsc/sc MEFs. Both double mutant and Fancd2–/– MEFs were equally sensitive to HMT+UVA. These experiments were repeated three times with identical results. Data points indicate mean±SEM for one experiment. At 3.0 Gy dose, P=0.001 (triple asterisk) for Fancd2–/–/Prkdcsc/sc versus Prkdcsc/sc. At 5.0 Gy dose, P<0.005 (double asterisk) for Fancd2–/–/Prkdcsc/sc versus Prkdcsc/sc.

 
The same cells were also exposed to increasing doses of IR in a similar cell growth assay. In agreement with previous results comparing the sensitivity of Fancd2 mutant MEFs and wild-type controls on a 129S4 background, Fancd2 mutant MEFs on the C57/Bl6J background were no more sensitive to IR than wild-type controls (Fig. 1B) (17Go). As expected for cells with impaired NHEJ, Prkdcsc/sc cells were more sensitive to IR than Fancd2–/– or wild-type controls. Interestingly, Fancd2–/–/Prkdcsc/sc fibroblasts grew worse than Prkdcsc/sc cells following DNA damage from IR.

Cells lacking a functional FA pathway display increased chromosome breaks and radials following treatment with ICLs (1Go). Radials form between non-homologous chromosomes, but are absent from sex chromosomes (38Go). We examined double mutant and control cells to determine whether cells lacking both NHEJ and Fancd2 had an increase in the number of radials following treatment with IR. Fancd2–/– cells showed a slight increase in radial formation over wild-type control cells, whereas Prkdcsc/sc cells had significantly more radials than Fancd2–/– cells (Table 1). The percentage of double mutant cells that contained at least one radial per cell was nearly double the percentage observed for Prkdcsc/sc cells following treatment with 5 Gy of IR. In addition, double mutant cells had a greater number of total radials per cell.


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  		Table 1. Radial formation in cells following treatment with IR
 
To further investigate the role of Fancd2 in the response to IR, we next exposed 6- to 8-week-old Fancd2–/–/Prkdcsc/sc mice and sex-matched Prkdcsc/sc littermate controls to 4.2 Gy whole body irradiation (Fig. 2). We have previously described a modest in vivo radiosensitivity phenotype in Fancd2 mutant mice in the mixed 129S4;C57/Bl6J background in which the LD50 for Fancd2 mutant mice (LD50=9.5 Gy) was reduced when compared with controls (LD50=11 Gy) (17Go). Fancd2–/–/Prkdcsc/sc mice on a C57/Bl6J background had a markedly reduced survival following irradiation when compared with Prkdcsc/sc controls. Only four of 17 Prkdcsc/sc controls died following 4.2 Gy of whole body irradiation, whereas three of three Fancd2–/–/Prkdcsc/sc mice died within 10 days after receiving the same dose. This finding, together with the in vitro cell growth assays, establishes the important role of Fancd2 in the response to IR-induced DNA damage and demonstrates that Fancd2 functions in a repair pathway that is distinct from NHEJ.



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  		Figure 2. In vivo IR sensitivity of Fancd2–/–/Prkdcsc/sc mice. Fancd2–/–/Prkdcsc/sc and littermate control Prkdcsc/sc mice were irradiated with 4.2 Gy of whole body irradiation. Double mutant mice were significantly more sensitive than Prkdcsc/sc controls.

 
Fancd2 functions in repair of restriction endonuclease-induced DSBs
The hypersensitivity of double mutant MEFs to IR suggests that Fancd2 functions in repair of IR-induced DNA damage. Because IR induces various types of DNA damage, we sought to test whether Fancd2 functions specifically in repair of IR-induced chromosomal DSBs. We measured the colony forming ability of SV40 immortalized Fancd2–/–/Prkdcsc/sc MEFs and Fancd2–/–/Prkdcsc/sc MEFs retrovirally corrected with Fancd2 cDNA following electroporation of the blunt-cutting restriction endonuclease, PvuII. Complemented cells were no longer sensitive to MMC-induced ICLs as measured by a cell growth assay (data not shown).

Restriction endonucleases have been previously shown to induce chromosomal DSBs, resulting in reduced cell viability (39Go–43Go). Double mutant cells had a reduced colony forming ability relative to Fancd2 corrected controls following electroporation of 5, 10 or 20 units of PvuII (Fig. 3). Heat-inactivated PvuII resulted in a colony forming ability comparable to mock electroporated cells (data not shown). Our results are in agreement with previous reports demonstrating a role of the FA pathway in response to restriction endonuclease-induced DSBs (20Go).



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  		Figure 3. Fancd2 functions in repair of PvuII-induced DSBs. Immortalized Fancd2–/–/Prkdcsc/sc (open circles) and Fancd2 corrected Fancd2–/–/Prkdcsc/sc cells (closed circles) were electroporated with 0, 5, 10 or 20 units of PvuII and plated. Each data point represents the combined mean±SEM for three independent experiments performed in duplicate (triple asterisk indicates P<0.001).

 
To control for potential variability in protein uptake between cell lines, both genotypes were electroporated with recombinant GFP and analyzed by FACS. The level of GFP positive cells relative to mock electroporated cells was approximately equal in both uncorrected double mutant cells (42.4%) and Fancd2 corrected double mutant cells (47.8%) (data not shown). Thus, the reduced colony forming ability of double mutant cells was not due to increased uptake of restriction endonuclease but rather due to reduced repair of chromosomal DSBs induced by the restriction endonuclease.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated that Fancd2 functions in a pathway for the repair of IR-induced DNA damage both in vitro and in vivo. Furthermore, the reduced colony forming ability of Fancd2–/–/Prkdcsc/sc cells relative to Fancd2 corrected controls following electroporation of a blunt-cutting restriction endonuclease demonstrates that Fancd2 functions specifically in the repair of chromosomal DSBs as opposed to other types of damage induced by IR. In previous reports, we were not able to demonstrate a differential sensitivity to IR between fibroblast derived from FA mutant mice and controls (15Go,17Go). In addition, the in vivo sensitivity of Fancd2 and Fancc mutant mice was only moderately increased relative to mutants, such as scid mice, that have a pronounced sensitivity to IR. The lower radiosensitivity of Fancd2 mutants compared with scid mice suggests that the FA pathway is less important in the repair of IR damage than NHEJ. This finding is consistent with previous observations that multiple DSB repair pathways exist in mammalian cells and that different species have varying dependencies on each pathway (44Go).

As NHEJ is the major DSB repair pathway in mammalian cells, we hypothesized that a role of the FA pathway might be only appreciable in vivo in the absence of a functional NHEJ repair pathway. Indeed, we observed an increased sensitivity to IR-induced damage for Fancd2–/–/Prkdcsc/sc mice and cells when compared with Prkdcsc/sc mice and cells. A number of recent experiments using reporter-based repair substrates have suggested that Fancd2 functions in HDR of DSBs (32Go,33Go,35Go). In addition, previous reports using plasmid-based studies have suggested that an end joining defect in FA nuclear extracts is distinct from NHEJ (19Go). The experiments described in this article are to our knowledge the first that have taken a genetic approach to demonstrate that Fancd2 is non-epistatic with Prkdc in vivo for repair of DSBs.

In addition to demonstrating a role of the FA pathway in repair of IR-induced DSBs, we have shown that NHEJ plays little to no role in the repair of HMT+UVA-induced ICLs. This finding is consistent with models of ICL repair that invoke multiple repair pathways, but not NHEJ, in the processing of ICLs (45Go,46Go). Although the precise steps of ICL repair in mammalian cells are yet to be fully understood, genetic and molecular biological studies have implicated XPF/ERCC1, a component of the nucleotide excision repair pathway, in the incision and unhooking of ICLs. A recombination step or translesion synthesis (TLS) is then predicted to complete the repair of ICLs (46Go,47Go). Recent experiments from our laboratory have investigated the details of ICL repair and show that ICLs are incised throughout the cell cycle but processed to a DSB intermediate only during the S-phase (48Go). Incision at ICLs is independent of FA pathway activation. However, the presence of active FANCD2 correlates with the appearance of DSBs, suggesting that FANCD2 may function in this later step of ICL repair.

The upstream FA protein, FANCC, has been suggested to act in both HDR and TLS for the repair or ICLs in chicken DT40 cells (47Go). Although our data cannot rule out a role of the FA pathway in TLS, the reduced colony forming efficiency in cells lacking Fancd2 following electroporation of PvuII supports a role of the protein in HDR of DSBs because blunt ended DSBs are not expected to be repaired by TLS. On the basis of our demonstration that Fancd2 functions in repair of a subset of DSBs, it is intriguing to speculate that the specific defect in ICL repair observed in FA cells involves an inability of FANCD2 to process the DSB intermediates that are generated during repair of ICLs.

In addition to variations among species in their relative use of HR versus NHEJ in the repair of DSBs, variations in the activity of each throughout the cell cycle have been documented. In chicken DT40 cells, NHEJ plays a major role in G1 and early S, whereas recombinational repair is preferentially used in late S/G2-phase (49Go). Activation of Fancd2 and its co- localizaton with HR proteins in nuclear foci occur specifically in S-phase of the cell cycle in wild-type cells (14Go). It will be interesting to compare the sensitivity of Fancd2–/–/Prkdcsc/sc cells and Fancd2 complemented double mutant controls to IR at various times during the cell cycle. One prediction is that double mutant cells irradiated during G1-phase of the cell cycle will not be more sensitive than complemented control cells. In contrast, double mutant cells may be more sensitive than complemented control cells when irradiated during S-phase of the cell cycle when the FA pathway is most active.

In these studies, we have used primary fibroblasts and whole animals to compare the relative radiosensitivity of Fancd2–/– and wild-type cells. Although we did not observe a difference in primary cells, we did observe a difference in whole animals, suggesting that specific cell types, such as hematopoietic stem cells, may have a greater dependence on the FA pathway for repair of DSBs. It is attractive to speculate that the FA pathway may function specifically in long-lived progenitors or stem cells and these cells, when deficient in the FA pathway, may acquire the genetic mutations required to progress towards cancer. Regardless of the precise nature of the defect in FA cells, the role of Fancd2 in repair of spontaneously generated DSBs may explain the genomic instability and cancer predisposition observed in human FA patients and Fancd2 knockout mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal husbandry
Fancd2+/– on a C57/Bl6J background (17Go) was crossed to Prkdcsc/sc (B6.CB17-Prkdcsc/SzJ) purchased from The Jackson Laboratory (Bar Harbor, ME, USA) to generate Fancd2+/–/Prkdc+/sc breeders. Genotyping was performed for Fancd2 mice (17Go) and Prkdcsc mice (29Go,50Go) as previously described. Fancd2+/–/Prkdc+/sc breeders were intercrossed to generate Fancd2+/–/Prkdcsc/sc breeders that were crossed to generate Fancd2–/–/Prkdcsc/sc and Prkdcsc/sc animals. Animals were maintained according to an approved IACUC protocol in the Department of Animal Care at Oregon Health and Science University.

Cell line generation
Primary MEFs were prepared from 4-week-old mice by digestion of tissue in collagenase/dispase (Sigma, St. Louis, MO, USA) for 1 h and collagenase (Sigma) for 1 h at 37°C. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 15% fetal bovine serum (FBS) (HyClone) and 1x penicillin/streptomycin (P/S) (Invitrogen, Carlsbad, CA, USA). Cells were immortalized by transfection with a plasmid expressing SV40 T antigen and passaged (51Go). Immortalized cell lines were grown in DMEM supplemented with 10% bovine calf serum (HyClone) and 1x P/S. Immortalized cells were complemented by infecting with pMMP-puro-mFancd2 retrovirus or pMMP-puro-empty retrovirus and selecting in 3.0 µg/ml puromycin as previously described (52Go–54Go).

DNA damage assays
Primary (passage 2) and immortalized cells were seeded in quadruplicate at a density of 500 cells per well in 96-well plates. Cells were treated with increasing doses of mitomycin C (MMC) (Sigma) or HMT+UVA (Sigma), as previously described (55Go). IR was administered at a dose of 142.5 rad/min from a 137Cs source. Following treatment, cells were allowed to grow at 37°C/5% CO2 for 5 days. Plates were frozen at –80°C. CyQuant (Molecular Probes, Eugene, OR, USA) was used to measure total DNA content as an indication of cell number as previously described (17Go).

Whole body irradiation
Fancd2–/–/Prkdcsc/sc and Prkdcsc/sc littermate controls were irradiated with 420 cGy at a dose of 142.5 cGy/min from a 137Cs source between 6 and 8 weeks of age. Following irradiation, animals were monitored daily, weighed and sacrificed if they appeared moribund. Survival curves were generated using Prism software (GraphPad Software, Inc., www.graphpad.com). Statistical significance between genotypes was determined using built-in analysis for survival curves consisting of a log-rank test yielding a P-value.

Restriction endonuclease electroporation
104 MEFs of the indicated genotypes were resuspended in 200 µl serum free DMEM and combined with 5, 10 or 20 units (50 µl) of PvuII (Roche, Indianapolis, IN, USA) diluted in the appropriate 1xbuffer/PBS. PvuII was heat inactivated after dilution in appropriate 1xbuffer/PBS for 18 h at 80°C. The mixture was electroporated in a 0.4 cm cuvette using a BioRad Gene Pulser (BioRad, Hercules, CA, USA) at a field strength of 0.75 kV/cm and 960 µF. Following electroporation, 1000 cells were plated on 100 mm plate in duplicate in DMEM+10% FBS. Cells were grown for 10 days and colonies were stained with a 1% Methylene blue solution (6.25% EtOH). The number of colonies from electroporation with 5, 10 or 20 units of enzyme was compared with the number of colonies from electroporation with 0 units of enzyme.

Green fluorescent protein electroporation/FACS
Twenty microgram of recombinant GFP (BD Biosciences Clontech, Mountain View, CA, USA) was electroporated into 2x10–6 MEFs using the conditions described earlier. Cells were washed in DMEM and resuspended in PBS containing 10 µg/ml propidium iodide. Analysis of mock electroporated and recombinant GFP electroporated MEFs was performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Cytogenetics
MEFs were plated in T-25 flasks in DMEM+10% FBS and treated with IR. After 16 h, cells were treated with 0.05 µg/ml colcemid (Gibco/BRL) for 4 h. Cells were digested with trypsin, placed in hypotonic medium consisting of 5% FCS and 75 mM KCl and fixed to slides. Slides were stained with Wright's stain (Fisher Scientific) for 3 min. Cells were observed using a Nikon E800 fluorescence microscope and captured using CytoVision software from Applied Imaging.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge Craig Dorrell PhD for assistance with FACS. M.G. was supported by NHLBI Program Project grant 1PO1HL48546 and S.H. was supported by a training grant appointment NIH 5 T32 GM008617-08.

Conflicts of Interest statement. The authors have no conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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