PMC

(A) GCR rate of either the wild-type (WT) or bud16Δ strain at ChrV-L.
(B) Whole-chromosome PFGE analysis of wild-type (W), parental bud16Δ (B), or bud16Δ strains that have undergone a GCR event (1–8). Left panel: ethidium bromide–stained gel. Right panel: Southern blot with a ChrV-specific probe (MCM3).

(A) Tetrad analysis of tpn1Δ (square) crossed to sno1snz1Δ (circle). Viable triple mutant tpn1Δ sno1snz1Δ can be recovered (circle in square).
(B) GCR rates at ChrXV-L of the indicated strains.
(C) Quantitation of Rad52-YFP foci per cell in wild-type and tpn1Δ cells.
(D) GCR rates at ChrXV-L of tpn1Δ sno1snz1Δ triple mutant grown with pyridoxine (+PYR) or without added pyridoxine to the media.
(E) Relative PLP levels of the tpn1Δ sno1snz1Δ triple mutant grown with pyridoxine (+PYR) or without added pyridoxine to the media.

(A) HeLa cells display elevated levels of 53BP1 foci following inhibition of Pdxk by 4-DP.
(B) Quantitation of cells with 53BP1 foci following 4-DP treatment.
(C) 4-DP treatment activates the DNA damage checkpoint in HeLa cells as measured by Chk2 phospho-Thr68 and γ-H2AX immunoblotting.
(D) SiHa cells treated with 4-DP accumulate γ-H2AX in S-phase, as measured by flow cytometry.

(A) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the wild-type (W), parent bud16Δrad52Δ strains (BR), or bud16Δrad52Δ strains that have undergone a GCR event (1–8). Asterisk indicates incomplete digestion products of ChrXV. An undigested Chromosome XV likely explains the presence of a signal in the well of strain 8, although a weak TRF signal can be detected.
(B) Quantitation of the terminal restriction fragment length decrease following GCRs when the PmeI TRFs from bud16Δ and bud16Δrad52Δ cells are compared to those of wild type.
TRF, terminal restriction fragment.

(A) GCR rates at ChrXV-L of the indicated strains. The data is presented as the fold-increase over the wild-type GCR rate +/− strandard error of the mean. Asterisk refers to genes with high GCR rates that were not identified in the screen, but were measured during the course of this study.
(B) A plasmid encoding BUD16 suppresses the high GCR rate of bud16Δ.
(C) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the wild-type (W), parent bud16Δ strains (B), or bud16Δ strains that have undergone a GCR event (1–8). Asterisk indicates incomplete digestion products of ChrXV.
(D) The bud16Δ mutation is synthetic lethal with the deletions of SNO1 and SNZ1.

(A) The intersection of vitamin B6 and dTMP biosynthesis. (i) Dietary vitamin B6 (i.e., pyridoxine) is imported into the cell via the Tpn1 transporter (S. cerevisiae gene names in brackets). (ii) Pdxk phosphorylates the B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) to generate PLP, which acts (iii) as a cofactor for serine hydroxymethyl transferase (SHMT). (iv) SHMT is necessary for the formation of methylenetetrahydrofolate (CH₂=THF), the methyl group donor for the conversion of dUMP into dTMP. (v) A deficiency in PLP is predicted to reduce dTMP levels leading to a nucleotide pool imbalance and incorporation of uracil into DNA.
(B) bud16Δ cells accumulate uracil in DNA, as measured by a modified aldehydic slot blot assay. The results of the slot blot (bottom panel) were quantified and shown in the graph. IDV refers to the integrated density values of the bands.
(C) Uracil accumulation in the bud16Δ genomic DNA is comparable to that observed in ung1Δ DNA.
(D) bud16Δ cells are sensitive to nucleotide depletion by hydroxyurea at 0.2 M. The results of a colony forming assay can also be found in .

(A) Tetrad analysis of crosses between bud16Δ and rad52Δ, rad51Δ, mre11Δ, or rad6Δ.
(B) Micrographs of wild-type (WT) or bud16Δ cells expressing Rad52-YFP. Left panels: differential interference contrast (DIC). Right panels: YFP fluorescence microscopy (YFP). Arrowheads point to Rad52-YFP foci.
(C) Quantitation of Rad52-YFP foci per cell. Three independent isolates were examined with over 180 cells per isolate counted.
(D) Survival curves of wild-type, bud16Δ or rad52Δ cells serially diluted and plated onto rich media +/− MMS (as indicated) and grown for 3–8 d at 30 °C in triplicate. Percent survival at a given MMS concentration represents the number of colony-forming units of the indicated strain divided by the colony-forming units of the wild type plated on media lacking MMS. No viable rad52Δ colonies were recovered at concentrations above 0.015% MMS.
(E) bud16Δ cells engage the DNA damage checkpoint. Upper panel: Rad53 immunoblots of extracts of the indicated strains. Middle panel: Ponceau stain for loading control. Lower panel: Rad53 activity assessed by auto-kinase assays []. The immunoblot and auto-kinase assays were performed on the same extracts.

(A) Schematic of the ChrXV-L GCR reporter chromosome. A GCR event in this region of ChrXV-L can result in the loss of the URA3 and CAN1 genes, which yields a canavanine and 5-FOA resistant strain (can^R 5-FOA^R). PSF3 is the first essential gene on ChrXV-L. HRI and HRII denote two regions of homology that are centered around the PAU20 and HXT11 genes, respectively.
(B) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the parent strain (W) or strains that have undergone a GCR event (1–9). Asterisk indicates incomplete digestion products of ChrXV.
(C) Array-based comparative genome hybridization of two strains that have undergone a GCR event at ChrXV-L. The above panel is a histogram representation of log₂-tranformed relative signal enrichments on Chromosome XV viewed in the University of California Santa Cruz genome browser []. The location of the array probes (reporters) is also indicated. Note the large loss of sequences on the left arm of ChrXV in both strains. The lower panel zooms to the ChrXV-L subtelomeric region. The breakpoint in strain (i) must be in the vicinity of the PAU20 gene (YOL161C), whereas that of strain (ii) resides in the vicinity of HXT11.