Figure 4

rPolθ-helicase presents ATP-dependent helicase activity. (A) Schematic representation of the helicase assay. Partial double-stranded DNA was composed of one oligonucleotide labeled with digoxigenin and another labeled with biotin; the dsDNA contained a 3′ tail to initiate unwinding. DNA was bound to a NeutrAvidin-sensitized plate via its biotinylated strand. After incubation with recombinant protein, the helicase activity was measured by analyzing the presence of strands labeled with digoxigenin using an anti-digoxigenin antibody. The absence of a signal indicated helicase activity. (B) The helicase assay was performed with only DNA or DNA and ATP (pink box – negative control: no helicase activity – detection of digoxigenin), with no DNA (purple box – no detection of digoxigenin), and in the presence of DNA and rPolθ-helicase in the presence or absence of ATP.