DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress

Abstract

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

Keywords: DNA polymerase; Fanconi anemia pathway; lesion bypass; whole genome fitness screens.

Fig. 1.

FA mutant synthetic lethality screen. (A) Schematic diagram depicting the gRNA-mediated whole genome fitness screen. (B) Resulting SVD v matrix of processed quantile-normalized BAGEL essential scores (BF) of FA-deficient and wild-type screens. Color similarity in the matrix represents the direction and amount of signal that is contributing to the individual component. (C) Histogram of Z-transformed component 4 scores coming from matrix u singular values, representing genetic contribution to differences between the FA knockouts and wild-type samples shown in the v matrix above. Blue, Z < −3; pink, Z > 3. (D) Comparison of mean BF scores in the FANCL^−/− and FANCG^−/− screens versus wild-type screen, colored as in C.

Fig. 2.

Pol ι loss leads to synthetic lethality with FANCL/FANCG/FANCD2 deficiency. (A–C) Clonogenic efficiency of Pol ι shRNA-knockdown HCT116 FANCL^−/− cells, FANCG^−/− cells, and HeLa FANCL^−/− cells. (D and E) Clonogenic efficiency of Pol ι shRNA-knockdown GM16631 and GM16635 patient cells. (F–G) Clonogenic efficiency of Pol ι shRNA-knockdown HeLa FANCD2^−/− and 293A FANCD2^−/− cells. (H) Flow cytometry histogram of Annexin V-positive cells in HCT116 wild-type (WT), FANCL^−/−, and FANCG^−/− cells subjected to shRNA-knockdown of Pol ι. (I) Flow cytometry histogram of Annexin V-positive cells in GM16631 and GM16635 patient cells subjected to shRNA-knockdown of Pol ι. (J) Flow cytometry histogram of Annexin V-positive cells in HeLa WT and FANCL^−/− cells subjected to shRNA-knockdown of Pol ι.

Fig. 3.

Pol ι loss renders unique synthetic lethality with FA deficiency. (A) BF distribution of DNA polymerase subunits from the FA synthetic lethality screens (green dots) and the Avana set (425 screens form Q4 2018) of genome-wide fitness screens (black dots). (B–D) Clonogenicity of Pol ι, η, and κ gRNA knockdown in HCT116 wild-type cells and FANCL^−/− cells. (E–G) Cell proliferation of Pol ι, η, and κ gRNA knockdown in HCT116 wild-type cells and FANCL^−/− cells.

Fig. 4.

Pol ι loss leads to DNA damage in FANCL/FANCG deficiency cells. (A) γH2AX foci staining of Pol ι gRNA knockdown in HCT116 wild-type cells and FANCL or FANCG deficient cells (72 h post transduction). (Scale bar: 10 µm.) The dotted circles in the first lane indicate the nucleus of the square circled cells in the second lane. The third lane is the enlarged view of the square circled cells. The dotted circles in the third lane indicate the boundary of the nucleus. (B and C) Quantification of γH2AX nuclear fluorescent intensity in experiment shown in A. (D and F) Immunoblotting of γH2AX and phosphorylated ATR and ATM of Pol ι gRNA knockdown in HCT116 wild-type cells and FANCL-deficient cells and mutated patient cells (72 h posttransduction). (E) Immunoblotting of phosphorylated CHK1 and CHK2 of Pol ι gRNA knockdown in HCT116 wild-type cells and FANCL-deficient cells (72 h posttransduction).

Fig. 5.

Pol ι loss leads to DNA replication fork collapse in FANCL/FANCG deficiency cells. (A) Schematic of DNA fiber analysis. Red track, CIdU; green track, IdU. (Scale bar: 2 µm.) (B) Proportion of stalled forks in Pol ι gRNA knockdown HCT116 wild-type cells and FANCL^−/− cells. (C) Proportion of restarted forks in Pol ι gRNA knockdown HCT116 wild-type cells and FANCL^−/− cells. (D) Length frequency of IdU tracts after exposure to HU in Pol ι gRNA knockdown HCT116 wild-type cells and FANCL^−/− cells. (E) Distribution of IdU track lengths in Pol ι gRNA knockdown HCT116 wild-type cells and FANCL^−/− cells. (F) Proportion of stalled forks in Pol ι gRNA knockdown HCT116 wild-type cells and FANCG^−/− cells. (G) Proportion of restarted forks in Pol ι gRNA knockdown HCT116 wild-type cells and FANCG^−/− cells. (H) Length frequency of IdU tracts of replication forks after exposure to HU in Pol ι gRNA knockdown HCT116 wild-type cells and FANCG^−/− cells. (I) Distribution of IdU track lengths in Pol ι gRNA knockdown HCT116 wild-type cells and FANCG^−/− cells. More than two hundred replication forks were analyzed for each sample.