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. 2013 Aug 23;41(21):9839–9847. doi: 10.1093/nar/gkt737

Figure 2.

Figure 2.

Low-energy CD spectra of 2AP-containing DNAs and hFEN1 WT- and mutant-DNA complexes. DNA constructs are illustrated schematically (red 2AP) with sequences shown in Supplementary Figure S1. (A) A dramatic divalent metal ion dependent reduction in 2AP exciton coupling signal occurred when product P−1−2 was bound to hFEN1, indicative of product unpairing. Unbound P−1−2 (blue), the corresponding single strand (CF−1−2, gray) and P−1−2 bound to hFEN1 (magenta) all in Ca2+ containing buffer. P−1−2 bound to hFEN1 in buffer containing 25 mM EDTA (green). (B) When product is not 5′-phosphorylated, (HO-P−1−2), there was no reduction in 2AP exciton coupling on addition of hFEN1- Ca2+. Unbound HO-P−1−2 (blue), the corresponding 2AP single strand (HO-CF−1−2, gray) and HO-P−1−2 bound to hFEN1 (magenta) all in Ca2+ containing buffer. HO-P−1−2 bound to hFEN1 in buffer containing 25 mM EDTA (green). (C) The presence of 5′-nuclease superfamily conserved K93 and R100 were required to reduce exciton coupling of P−1−2. Unbound P−1−2 (blue), P−1−2 bound to WT hFEN1 (magenta), Y40A (cyan), K93A (purple) and R100A (orange) in Ca2+ containing buffer. See Supplementary Figure S3A for spectra in EDTA buffer. (D) A similar dramatic divalent metal ion-dependent reduction in 2AP exciton coupling signal occurred when substrate S−1−2 was bound to hFEN1, indicative of substrate unpairing. Unbound S−1−2 (blue), the corresponding 2AP single strand (F−1−2, gray), and S−1−2 bound to WT hFEN1 (magenta) in Ca2+ buffer. S−1−2 bound to hFEN1 in buffer containing 25 mM EDTA (green). (E) The presence of 5′-nuclease superfamily conserved K93, R100 and Y40 were not required to reduce exciton coupling of S−1−2. Unbound S−1−2 (blue) and S−1−2 bound to WT hFEN1 (magneta), Y40A (cyan), K93A (purple), R100A (orange) all in Ca2+ containing buffer. (F) No changes in low energy CD signal occurred on complexation when 2APs were located away from the region of the substrate proposed to unpair. Unbound S−8−9 (blue), the corresponding single strand (F−8−9, gray) and S−8−9 bound to hFEN1 (magenta) all in Ca2+ containing buffer. S−8−9 bound to hFEN1 in buffer containing 25 mM EDTA (green). Each measurement was independently repeated and gave equivalent results.