Yeast Origins Establish a Strand Bias for Replicational Mutagenesis Youri I. Pavlov, Carol S. Newlon, Thomas A. Kunkel  Molecular Cell  Volume 10, Issue 1, Pages 207-213 (July 2002) DOI: 10.1016/S1097-2765(02)00567-1

Figure 1 The ura3-29 Missense-Mutation Reversion System for Detecting DNA Strand-Specific Mutations (A) Rates and proportions of the three types of revertants of the TCT codon arising spontaneously in wild-type, ogg1, and pol3-Y708A strains or induced by HAP (165 μM) in the wild-type strain. Strain 8C-YUNI101 strain was used (Pavlov et al., 2001b), and the corresponding mutants were obtained by transformation. Ratios of different genetic events were calculated based on 30–100 revertants sequenced in this and previous studies (Pavlov et al., 2001b; Shcherbakova and Pavlov, 1996). The numerical Ura+ entries are mutation rates except for the wt, HAP entry (marked by asterisk), which is a mutation frequency. (B) Mechanisms of mutation induction by strand-specific base analogs. Left, C·G to A·T transversions induced by GO in DNA. For round one, two replicating DNA strands with target TCT codons are shown. Misincorporation of dATP opposite GO occurs in only one strand (bold). For round two, replication of this strand (bold) leads to mutation fixation. Right, G·C to A·T transitions induced by dHAPTP. Designations are the same as in the left part. The misincorporation of dHAPTP initiates mutagenesis in the opposite DNA strand compared to GO mutagenesis. (C) Spontaneous and HAP-induced revertant yields in strains with two orientations of the ura3-29 reporter inserted into the BIK1 gene. Cells plated by a multiprong Poissoner device (Khromov-Borisov et al., 2002) were allowed to grow on YPD medium with or without HAP for 48 hr and were then replica plated onto complete minimal medium lacking uracil. (D) System for integrating the ura3-29 reporter cassette in yeast chromosome III. Plasmids are shown with the LEU2-ura3-29 cassette in two orientations. “f” is a forward primer with homology to pUC18 plasmid DNA (solid line) and with homology to a defined site of the W (Watson) strand of yeast chromosome III (dashed line) (see the Experimental Procedures for details). “r” is a reverse primer with homology to the reverse strand of the pUC18 and with distal region (dashed line) homologous to the reverse strand of the C (Crick) strand of yeast chromosome III. PCR fragments obtained with these primers were integrated into corresponding sites in the yeast chromosome. Molecular Cell 2002 10, 207-213DOI: (10.1016/S1097-2765(02)00567-1)

Figure 2 Patterns of HAP-Induced ura3-29 Reversion at Specific Locations in the 39 Kb Region of Chromosome III in Wild-Type and ARS-Deletion Strains (A) Map of the left arm of yeast chromosome III. Distances between elements are in thousands of base pairs and are shown above the chromosome. The region studied in this work is expanded below the chromosome with locations and names of insertion alleles. Replication origins are shown as black rectangles. (B) Ratio of reversion rate of the ura3-29 allele in orientation one to its rate of reversion in orientation two at different locations in chromosome III in the ogg1 strain. Black rectangles indicate two functional ARSs. Each bar represents the ratio of reversion rate in orientation one to orientation two at that location, which corresponds to the named position in (A). The scheme below represents the region of chromosome III undergoing bidirectional replication initiated at ARS305 and the ARS306. Continuous arrows represent newly synthesized leading-strand DNA while multiple arrows represent newly synthesized lagging-strand DNA. Replication forks move to the left and to the right from each origin. They meet in the middle of the region at a site that is equidistant from both origins. The encircled region indicates the reporter allele in orientation one showing dATP incorporation opposite GO into lagging-strand DNA. (C) As in (B), but ratio of HAP-induced reversion frequency of the ura3-29 allele in orientation one to its frequency of reversion in orientation two at different locations in chromosome III in the wild-type strain. The encircled region indicates the reporter allele in orientation one showing dHAPTP incorporation into leading-strand DNA. (D) Ratio of HAP-induced reversion in orientation one to reversion orientation two at different locations in chromosome III in strains carrying the deletion ΔARS305 (white triangle) and schematic representation of that region of chromosome III undergoing replication, proceeding to the left from ARS306. (E) As in (D), but for strains with the deletion ΔARS306 (white triangle). Replication forks move to the left from ARS307 and to the right from ARS305 and meet near the position of the ARS306 deletion. (F) As in (D), but for strains with the double deletion ΔARS305 ΔARS306. A replication fork originating from ARS307 goes from right to left through the entire region. Molecular Cell 2002 10, 207-213DOI: (10.1016/S1097-2765(02)00567-1)