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.