FIGURE 3.

FAN1 can also traverse a cross-link on a linear DNA substrate. A, schematic of the linear DNA substrates labeled at the 3′ terminus of the upper strand by fill-in reactions with [α-³²P]dTTP. A schematic of the expected products is shown next to the corresponding substrates (a, b, and a'–c'). Red asterisks indicate the positions of the ³²P-labeled nucleotides. Fragments invisible on the autoradiograph are shown in gray. B–D, 5′ to 3′ exonuclease activities of FAN1 (B), EXO1 (C), and FEN1 (D) on the indicated substrates. Reaction conditions and protein concentrations were as in Fig. 2. Lowercase letters correspond to the products in A. M, low molecular weight marker (Affymetrix). The oligonucleotide sizes are indicated. The black asterisk indicates the position of the non-cross-linked 28-mer oligonucleotide that was present in small amounts in the ICL substrate. Bottom panels, quantifications of the degradation fragments. Error bars show mean ± S.D. (n = 3). E, comparative analysis of cleavage products generated on the 3′ side of the cross-link by the three nucleases as well as by the nuclease-dead mutants of FAN1 (D960A) and EXO1 (D173N). The reaction conditions were as in Fig. 1E. F, FAN1 WT and D960A activity on a recessed linear DNA substrate shown schematically above. Concentrations were as indicated, and samples were withdrawn after 1, 10, 30, or 60 min (WT, lanes 2–5) or after 1, 30, and 60 min (D960A, lanes 6–8). For the substrate sequence, see “Experimental Procedures.” Quantifications are as indicated above. Black asterisks indicate the non-cross-linked oligonucleotides present in the substrate preparation. The dashed line represents missing lanes.