Figure 4. Bulky Lesions on the 5′ Overhang Strand Impede the Core7 Helicase Activity.

(A) DNA unwinding activities of Core7 on a normal 5′ overhang DNA (N_45/35), with Cy5 on the top strand (Cy5_TOP), a mismatched base pair (M_45/35), or Cy5 on the bottom strand (Cy5_BOT). DNA substrates are diagramed at the bottom of each panel, and asterisks indicate 5′-³²P labels. Core7 concentrations were 10, 20, 40, and 80 nM in each assay. Triangles (Δ) indicate the heat-denatured substrates, and the “C” stands for control of DNA substrate alone.

(B) Effects of CisPt on the Core7 helicase activity were compared by placing the lesion CisPt on the top (Pt_TOP) or the bottom strand (Pt_BOT) of the normal DNA duplex N_44/34 or N_54/44, respectively, each with a 5′ overhang (Table S2).

(C) DNA unwinding activities of Core7 with an AP lesion on the top (AP_TOP) or bottom strand (AP_BOT).

(D) Unwinding of DNA with a 3′ overhang by Core7 was undetectable. The Cy5 near the ds-ss junction promoted DNA unwinding, perhaps by melting DNA locally and generating a free 5′ end.

(E) The XPD^KR mutant Core7 had very weak helicase activity and was inhibited by CisPt present on the strand scanned by XPD. To detect the weak activity, protein concentrations were increased to 80–320 nM.

(F) The XPB^KR mutant Core7 had slightly reduced helicase activity compared with WT.

(G) Relative activities of WT Core7 in unwinding DNAs with a 5′ overhang. Helicase activities (%) were normalized to the corresponding undamaged DNA substrates and plotted in the same colors as DNA diagrams shown in (A)–(C). Standard deviations were estimated based on triplicates.