Figure 5.

Pol μ misincorporation analysis at gapped-DNA intermediates containing position 74. DNA substrates in which position 74 is embedded in gaps of 1 nt (A) or 2 nt (B) were obtained as described in Materials and Methods. Labeled oligonucleotide primers are shown in gray letters. The WRCY hotspot motif sequence containing position 74 (arrow) is indicated with white letters. Templating residues available at each step are indicated with white letters on black background. Polymerization assays were carried out as described in Materials and Methods, in the presence of the indicated concentrations of each individual dNTP. After 30 min at 30°C, primer extension was analyzed by 8 M urea/PAGE and autoradiography. Mobility of the unextended primer is indicated with an asterisk at the left of the autoradiograph. A scheme detailing the different stages of the slippage-mediated dislocation model that explains the main misincorporation is shown (see text for further details). Transiently misaligned nucleotides generated by dislocation or strand displacement are indicated with white letters on black circles. Mismatched nucleotides are indicated with unfilled letters.