FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair

Abstract

Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes to MMR in vivo; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

Keywords: DNA repair; EXO1; FAN1; MLH1; MSH6; exonuclease; mismatch repair; mutational signature.

FIG 1

Recombinant FAN1 or EXO1 rescue the MMR defect in extracts of EXO1^−/− or EXO1^−/− FAN1^−/− chicken B-lymphocyte DT40 cells. (A) Scheme of the substrate used in the in vitro MMR assays. The T/G mismatch at position 46 of the phagemid heteroduplex makes it refractory to AclII cleavage at this site. Digestion with AclII generates two fragments, of 2,823 and 373 bp (panel B, right scheme). Repair of the Nt.BstNBI-nicked T/G substrate to T/A regenerates the AclII cleavage site. AclII digest thus yields two additional bands, of 1,516 and 1,307 bp (panel B, left scheme). Repair efficiency was defined as the ratio of the intensities of the 1,516-bp versus the 2,823-bp band in GelRed-stained agarose gels. (B) MMR assay with DT40 nuclear (NE) and cytoplasmic (CE) extracts. MMR-proficient HEK293T-MutLα(+) nuclear extract was used as the positive control (293). Incubation of the substrate in MMR buffer without extract was used as the negative control (–). (C) EXO1^−/− DT40 cell extracts are largely MMR-deficient. (D) FAN1^−/− DT40 extracts are MMR-proficient, unlike extracts of MLH1^−/− DT40 cells. (E) MMR in EXO1-deficient DT40 extracts can be rescued by recombinant human EXO1 (hEXO1, 400 ng [++]) and chicken FAN1 (gFAN1, 80 ng [+] or 160 ng [++]) but not the nuclease-dead gFAN1 D977A (160 ng [++]) variant. The bottom panel is an autoradiograph of the same gel. (F) Recombinant hEXO1 (400 ng [++]) and gFAN1 (80 ng [+]) can rescue MMR in FAN1^−/− EXO1^−/− DT40 extracts. Panels B to F show images of representative GelRed-stained 1% agarose gels run at 200 V in TAE buffer.

FIG 2

The ability of FAN1 to rescue the MMR defect in human cells lacking EXO1 or both EXO1 and FAN1 is affected by FAN1 interaction with MLH1. (A) Alignment of the putative MLH1 interaction motifs in human (h) and chicken (g) FAN1 and PMS2. The conserved leucines that were mutated to alanines are marked with asterisks. (B) WT FAN1 and its LALA variant have comparable activities in a nonspecific nuclease assay, which measures the relaxation and cleavage of a supercoiled plasmid substrate. The FAN1 DA nuclease-dead mutant was included as the negative control. Oc, open circular; lin, linear; sc, supercoiled plasmid. (C) WT EXO1 or FAN1, but not the respective nuclease-dead DA mutants, can rescue the MMR activity of HEK293 extracts in which EXO1 was depleted with siRNA (Fig. S2A). The activity of the LALA variant in this MMR assay was diminished compared to the WT protein. (D) Quantitation of the time course shown in panel C. (E) Recombinant human FAN1 or EXO1, but not FEN1, rescues the MMR defect in nuclear extracts of FAN1^−/− EXO1^−/− TK6 cells. (F) Comparison of the ability of equal amounts of WT FAN1 and its LALA variant to complement the in vitro MMR deficiency of FAN1/EXO1-deficient TK6 cells. Panels B, C, E, and F show representative images of GelRed-stained 1% agarose gels run at 200V in TAE buffer.

FIG 3

FAN1 contributes to MMR efficiency in vivo. The indicated TK6 cell lines were cotransfected with an EGFP control plasmid and an equal amount of a reporter plasmid containing a T/G mismatch in the mCherry gene, as well as a nick in the T-strand. MMR efficiency was estimated from the ratio of the EGFP (green) and the mCherry (red) signal generated by correction of the T/G mismatch to C/G, which converts a TAG stop codon in the mCherry ORF to a TGG Trp codon. The MMR efficiency of the mutants is shown relative to WT cells, which were arbitrarily set to 100%. The results of three independent experiments are shown.

FIG 4

FAN1 deficiency augments the resistance of EXO1-deficient TK6 and HAP1 cells to MNU and TG. (A) Clonogenic assay showing the response of WT, FAN1^−/−, EXO1^−/−, and FAN1^−/− EXO1^−/− TK6 cells to MNU. (B and C) MTT assays showing the response of WT, FAN1⁻, EXO1⁻, EXO1⁻ FAN1⁻ and MSH6⁻ HAP1 cells to MNU (B) and TG (C). The results of three independent experiments each carried out in triplicate are shown. Error bars represent the standard error of the mean.

FIG 5

Mutational signatures of WT, FAN1^−/−, EXO1^−/−, and FAN1^−/− EXO1^−/− TK6 cells. (A) Scheme of the experiment. After generation of the knockout cell lines, single cell clones of each genotype were isolated and expanded. A portion of these cells was used to isolate DNA for sequencing (p0), and the remainder was grown for 40 cell divisions (p40) to permit mutation accumulation. Three single clones from each of the p40 populations were then picked and expanded, and their DNAs were subjected to whole-genome sequencing. (B) Summary of substitution mutations in the indicated cell lines. (C) Summary of insertion and deletion mutations in the indicated cell lines. [±1Rep], insertion/deletion of a single nucleotide in a mononucleotide repeat; [>±1Rep], insertion/deletion of more than one nucleotide in a mononucleotide repeat; Mh, microhomology-directed insertion/deletion. The columns represent an average mutation count in three independent clones.