DNA binding activity of Rep68 and TrwC/Rep.

(A) Rep68 and TrwC/Rep DNA substrates used for binding assays. Rep68 substrates were an AAV ori heteroduplex, and an equivalent AAV ori substrate mutated in the RBS sequence (RBSmut). trs is shown in boldface; nicking site is indicated by a slash. RBS is highlighted in bold italic. The AAVS1 minimal sequence (30) is presented below. TrwC/Rep chimera substrates were oligonucleotides oriTw(25+8) and oriTw(25+8) mutated in the inverted repeat IR (IRmut). Horizontal lines and nucleotides highlighted in boldface show sequence requirements for binding and nicking activities [77]. The nic site is represented by a slash. The IR recognized during binding is indicated with arrows; distal and proximal arms are shown. * represents the radioactively labelled strand. (B) and (C) EMSA assays with His-Rep68 and His-TrwC/Rep chimera, respectively. 30 fmol of the indicated radiolabelled substrates were incubated either with 100 ng of His-Rep68 or 200 ng of His-TrwC/Rep. All reactions contain 400 ng of poly(dI-dC) as nonspecific DNA. Competition assays were done using cold competitor DNA at 10- to 90-fold molar excess. Products were analysed on a native 6% polyacrylamide gel. Percentage of bound substrate (“% shift”) was calculated using Image Quant TL software. Bound and unbound products of the reaction are indicated with arrows. The DNA binding percentage is a representative example obtained from 3 independent experiments (n = 3). (D) Fluorescence polarization assay with oriTw(25+8) DNA labelled with carboxyfluorescein at 5 mM concentration. Binding was performed in 25 mM HEPES (pH 7.0), 200 mM NaCl at room temperature. Data analysis was performed as described by Yoon-Robarts et al. [57].