Fig 1.
Structures of model DNA used in the study.
For oligonucleotide sequences see S1 Table.
Fig 2.
RPA binding to DNA containing a flap with a gap (A, B) or a gap only (C).
The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-32P-labeled DNA and RPA at the indicated concentrations. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. (A) Increasing amounts of RPA were added to DNA containing a 26 nt gap and a flap. (B) RPA was added to DNA containing a 10 nt gap and a flap. (C) RPA was added to DNA containing only gaps: a 10 nt gap (lanes 1–11) or a 26 nt gap (lanes 12–22). (D) Summary plot of the data of the binding experiments shown in panels A-C. Averages and standard deviations were estimated from three independent experiments.
Fig 3.
RPA localization on DNA structures containing a damaged flap and a 26 nt gap (A) or a 10 nt gap (B).
The reaction mixtures (20 μl) contained buffer A, 10 nM 5′-32P-labeled photoreactive DNA containing 5I-dUMP within gap (lanes 1–10) or flap (lanes 11–20) and RPA at the indicated concentrations. A schematic view of the DNA structures is presented: a triangle indicates the position of the bulky lesion, dashes and numbers indicate the position of 5I-dUMP relative to the start (ss/ds DNA junction). The photocrosslinking products were separated by SDS-PAGE and visualized by autoradiography. (C) Quantitative analysis of the data from (A) and (B) for the p70 subunit. Averages and standard deviations were estimated from three independent experiments. The bottom panel presents a schematic view of the RPA localization on the DNA based on maximum photocrosslinking intensity.
Fig 4.
XPA binding to DNA containing a 10 or 26 nt gap (A) or both a gap and a flap (B). (C) XPA binds bubble-DNA more effectively than flap-gap containing DNA.
Averages and standard deviations were estimated from three independent experiments.
Fig 5.
Comparative analysis of RPA and XPA binding to DNA containing the 26 nt gap with the flap (A) or the 26 nt gap only (B).
The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-32P-labeled DNA and the indicated concentrations of proteins. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. White circles indicate putative RPA-XPA-DNA complexes with unknown stoichiometry.
Fig 6.
Comparative analysis of RPA and XPA binding to DNA containing a 10 nt gap with a flap (A) or a 10 nt gap only (B).
The reaction mixtures (10 μl) contained buffer A, 10 nM 5′-32P-labeled DNA and the indicated concentrations of proteins. A schematic view of the DNA structures is presented at the top: a triangle indicates the position of the bulky lesion. White circles indicate putative RPA-XPA-DNA complexes with unknown stoichiometry.
Fig 7.
RPA-XPA complex formation. Fluorescently labeled RPA was titrated by XPA in the absence of DNA or in the presence of unlabeled (A) or 32P-labeled (B) ssDNA.
The reaction mixtures (20 μl) contained buffer A, 10 nM Flu-RPA and indicated concentrations of XPA and/or DNA. A schematic view of the DNA structures is presented at the top: the triangle indicates the position of the bulky lesion. (A) The right panel shows the quantitative analysis of the data from the EMSA experiments. Percentage of the XPA protein active in DNA binding was 20%. Averages and standard deviations were estimated from three independent experiments.
Fig 8.
Stability of the RPA-XPA complex in the presence of DNA containing gap/flap-gap26 (A) or gap/flap-gap10 (B).
The reaction mixtures (20 μl) contained buffer A, 10 nM Flu-RPA, 40 nM XPA and the indicated concentrations of DNA. A schematic view of the DNA structures is presented at the top: the triangle indicates the position of the bulky lesion. The bands indicated as BSA-BPB correspond to a complex of BSA with bromophenol blue present in the loading buffer.
Fig 9.
Proposed location of the RPA and XPA proteins in the pre- and post-excision complexes.
After XPF-ERCC1 incision, RPA remains bound with the undamaged strand in the gap created. RPA can be translocated on the flap when gap is shortened during the re-synthesis step.