Fig. 5: DinB1 promotes −1 and +1 frameshift mutations in homo-oligonucleotide runs.

a leuD and kan frameshift (FS) reporter assays. leuD and kan open reading frame N-termini in which 2 base pairs in the second leuD codon were removed (blue box) or 4T/5T runs (red box) were incorporated upstream the start codon of kan. Reversion can occur by FS mutations that restore the leuD or kan reading frames resulting in phenotypic leucine prototrophy (leu⁺) or kanamycin resistance (kan^R). The red box in leuD shows the run of 3T in which the majority of detected FS in leu⁺ were found. b leu⁺ and c, d kan^R frequencies in the indicated strains in presence of inducer. Results shown are means (±SEM) of data obtained from biological replicates symbolized by gray dots. Stars above the means mark a statistical difference with the reference strain (empty) (*, P < 0.05; ***, P < 0.001). p-values were obtained on log-transformed data by one-way ANOVA with a Bonferroni post-test. Relative (pie chart) and absolute (bar chart) frequencies of nucleotide changes detected in leuD of leu⁺ cells or in kan of kan^R cells represented with colors: red = −1 FS in the b 3 T run, c 4 T run, or d 5 T run, other colors = FS outside of the run, and gray = no detected mutation. The number of sequenced leu⁺ or kan^R colonies is given in the center of each pie chart. e DinB1 polymerase reaction mixtures containing 5’ ³²P-labeled primer-template DNAs with A4, A6, A8, T4, T6, or T8 runs in the template strand (depicted below and included as indicated above the lanes) and 125 µM deoxynucleotides and dideoxynucleotides (as specified above the lanes) were incubated at 37 °C for 15 min. DinB1 was omitted from reactions in lanes –. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The +1 slippage products are indicated.