1. Volume 15, Issue 4, Pages 607-620 (August 2004)
The Fanconi Anaemia Gene FANCC Promotes Homologous Recombination and Error- Prone DNA Repair 
Wojciech Niedzwiedz, Georgina Mosedale, Mark Johnson, Chong Yi Ong, Paul Pace, Ketan J. Patel 
Molecular Cell 
Volume 15, Issue 4, Pages (August 2004)
DOI: /j.molcel

2. Figure 1
The Chicken ΔFANCC Displays Some Features that Are Common to Human FA Cells
(A) (Left) BRDU/Propidium Iodide cell cycle analysis of wild-type (wt) and FANCC KO (ΔFANCC) DT40 cells in log phase. The phases of the cell cycle are gated, and the proportion of cells in each phase is displayed as a percentage of cells analyzed. (Right) FANCD2 western blot of wt and ΔFANCC. Cells were irradiated with 10 Gy and allowed to recover for 2, 4, and 6 hr. Whole-cell lysates were then probed with anti-human FANCD2 antisera to detect chicken FANCD2 by Western blot. Upon irradiation FANCD2 migrates as two isoforms D2-L (monoubiquitinated) and D2-S (unmodified).
(B) (Top left) Chromosome breakage assay of wt and ΔFANCC cells exposed to a single dose of the DNA crosslinking agent MMC (50 ng/ml for 12 hr). Colony survival assay after exposure to X-irradiation (top right) and exposure to MMC (bottom right). Cell viability assay for exposure to cisplatin (bottom left). Standard errors are shown on graphs.
(C) (Top) The induction of recombination mediated repair of an inactive GFP coding substrate by the I-SceI endonuclease. This is displayed as the percentage of cells that become GFP positive as detected by FACS analyses of DT40-DRGFP, ΔFANCC-DRGFP, and ΔXRCC3-DRGFP cells with and without transfection with an I-SceI expression plasmid. Each bar represents a mean of four independent experiments. (Bottom) Measurement of the number of homologous integration events (Int) into two loci (Ovalbumin and XRCC2) in each of two ΔFANCC cell lines and wt DT40, as a ratio over the total number of drug resistant colonies (TDR).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
XRCC2 Ablation in ΔFANCC Cells Does Not Potentiate Sensitivity to DNA Crosslinking Agents but Has a Marked Impact on Cell Growth and Viability
(A) (Top left) FANCD2 Western blot to detect FANCD2 isotypes (L and S) of ΔFANCC, ΔXRCC2, and ΔFANCCΔXRCC2. Abbreviations: U, untreated; 6h, 6 hr recovery after 10 Gy of irradiation. (Right) Cell viability assay following exposure to cisplatin. (Bottom left) Chromosome breakage assay of untreated cells (−) and cells exposed to MMC (40 ng/ml for 12 hr) (+). For each data point 100 metaphases were scored blind for unambiguous aberrations.
(B) Table shows the cell doubling times, percentage of apoptotic cells present, and clonogenic survival in the DT40 strains listed. Cloning efficiency is expressed as a mean percentage value obtained from seven independent experiments. Fold reduction in plating efficiency against wt DT40 is as follows: ΔFANCC (2.9-fold), ΔXRCC2 (9.8-fold), ΔFANCCΔXRCC2 (460-fold), and ΔFANCCΔXRCC2 complemented with ChFANCC (7.4-fold). (Bottom) Spontaneous SCE events in wt DT40, ΔFANCC, ΔXRCC2, and ΔFANCCΔXRCC2 strains. For each cell line a total of 50 metaphases were scored blind for SCE events on chicken macro chromosomes.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
ΔFANCC Cell Lines Have Reduced Templated and Untemplated Changes in Response to Endogenously Generated Abasic Sites in the Ig V Gene Locus
(A) Schematic diagram outlines that the generation of IgM surface-negative variants occurs by either untemplated insertions (lollipop) or gene conversion (bar).
(B) sIgM+ to sIgM− fluctuation analysis of DT40, ΔFANCC (two clones), and one clone complemented back with chicken FANCC cDNA. Each point represents a single clone expanded for up to 1 month and then analyzed by FACS for loss of sIgM expression. For each cell line, 24 clones were expanded and the median percentage prevalence of sIgM negatives is determined. p values are calculated for statistical significance.
(C) 20 sequences containing sIgM inactivating gene conversion tracts are shown for DT40 and ΔFANCC cells. Black bars denote gene conversion tracts. Many sequences contain more than one tract. (Right) Pie chart indicates number of gene conversion tracts seen in individual sequences for both DT40 and ΔFANCC. Insets represent total number of sequences analyzed.
(D) (Top) sIgM positive to negative fluctuation analysis: (top left) in DT40, ΔXRCC3 (two clones) and ΔXRCC3/ΔFANCC (two clones); (top right) in DT40, ΔXRCC2, ΔXRCC2ΔFANCC, and ΔXRCC2ΔFANCC complemented back with chFANCC cDNA. p values are calculated for statistical significance. (Bottom) Pie charts of the number of untemplated mutations per individual sequence in sorted sIgM-negative cells for the double mutant strains. Insets represent total number of sequences analyzed.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
FANCC Functions in Rev1- and Rev3-Dependent DNA Crosslink Repair
(A) Sensitivity to cisplatin (cell viability assay) (left) of DT40, ΔFANCC, ΔRev1, or ΔRev3 single mutants compared to ΔFANCCΔRev1 or ΔFANCCΔRev3 (in each case two independently derived clones).
(B) Chromosome breakage assay of cells exposed to a range of doses of the DNA crosslinking agent cisplatin. The graph shows the standard error of the mean for total aberrations per metaphase for each experimental point. Very low doses were used because of the extreme sensitivity that ΔRev1 and ΔRev3 cells possess to crosslinks.
(C) FANCD2 Western blot on whole-cell lysates of DT40, ΔRev1, ΔRev1ΔFANCC, ΔRev3, and ΔRev3ΔFANCC following irradiation (+) or untreated (−).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Defective Induction of Sister Chromatid Exchange Events (SCE) in ΔFANCC, ΔRev1, and ΔRev3 Cells in Response to Crosslinking Agents, but Not in Response to Exposure to the Base-Damaging Agent 4NQO
(A) (Top) Schematic representation of the SCE experiment shown below. After harvest cells were stained for the visualization of differentially labeled chromosomes. (Below) Induction of sister chromatid exchange events in wt, ΔFANCC-, ΔRev1-, and ΔRev3-deficient cells after exposure to a range of doses of 4NQO (top row), MMC (middle row), and cisplatin (bottom row). For each bar 50 metaphases were scored blind for SCE events on macrochromosomes and the mean number calculated (error bars represent ±SEM).
(B) Images of SCE metaphases obtained from ΔRev1 and ΔRev3 cells exposed to MMC. Arrows mark chromosomes containing chromatid breaks. In most of these cases there appear to be no crossover events at these sites.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Colocalization of FANCD2 with Ectopically Expressed YFP-Rev1 in Nuclear Foci upon Replication Arrest in Human HeLa Cells
(A) HeLa cells were untreated (top row) or treated with X rays (10Gy) (second row), hydroxyurea (HU) at 5 mM for 7 hr (third row), or thymidine (THY) 2 mM for 24 hr (fourth row). Cells were fixed and stained for FANCD2 (red) and visualized for the presence of YFP-Rev1 (green). Colocalized proteins are seen as orange/yellow.
(B) FANCD2 monoubiquitination after thymidine block and release. HeLa cells were synchronized by using a double thymidine block. Release from block was then monitored for cyclin B expression, which increases as S phase progresses, culminating in a sharp decline at mitosis (M). At 0 hr most of the FANCD2 is monoubiquitinated (D2-L). This declines as S phase progresses up to G2 and M, when this isoform virtually disappears, leaving only unmodified FANCD2 (D2-S).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Models Outline a Combined FA/TLS/HR Process to Repair Crosslinks as Well as an Explanation for Raised SCE in Chicken FA Knockouts
(A) A Model for the potential mechanisms by which FA, TLS, and HR enzymes function to resolve DNA crosslinks in vertebrates. The initial unhooking of a crosslink does not require DNA replication; however, the subsequent steps require replication. In both instances, a stalled replication fork intiates repair that would involve translesion synthesis (TLS) and nucleotide excision repair (NER). Route 2, however, requires an HR step to complete repair. FANCC could function to initiate the repair response by stabilizing the stalled replication fork and by protecting the double-strand break from deletion prone repair pathways. Red lines mark repaired strands and open and closed rectangles indicate point mutations created by the TLS step.
(B) This model outlines a potential mechanism that may result in spontaneous elevated sister chromatid exchanges seen in FANCC and some TLS-deficient DT40 mutants. The thick arrow marks the route taken for the repair of replication blocking lesions in the absence of FA proteins or TLS.
Molecular Cell  , DOI: ( /j.molcel )