Figure 5.

RecQL4_N54 binds dsDNA without apparent sequence preference. (A) Electrophoretic mobility gel shift assay were performed with RecQL4_N54 (2 µg/30 µM) and DNA fragments (200 ng) covering the sites of DNA replication initiation of three well-characterized human origins as well as three control fragments from proximal sites. Details on the DNA fragments are provided in Supplementary Table S1. The DNA binding buffer was supplemented with 100 mM KCl where indicated. (B) Binding of DNA fragments derived from the LB2 origin of replication (42) by RecQL4 was evaluated by a DNA bead binding assay. Biotinylated dsDNA was pre-incubated with RecQL4 following binding to magnetic SA beads immobilized to magnetic columns. The flow through (FT), the first wash (W) and the elution (E) fractions were analysed by SDS–PAGE. RecQL4_N54 is detected in the elution fractions of all samples containing DNA indicative for DNA binding irrespective of fragment length. The protein band corresponding to RecQL4_N54 is indicated by an arrow. The additional band in the elution (denoted by an asterisk) is SA released from the magnetic bead during sample preparation for SDS–PAGE. Input represents purified RecQL4_N54. The positions of the molecular weight markers (in kDa) are indicated on the right.