The C terminus of Bur1 binds to RPA and is needed for resistance to transcription inhibitors and genotoxic agents. A, schematic of Bur1. The N-terminal half of Bur1 contains the conserved cyclin-dependent kinase domain, whereas the C-terminal half is not conserved. B, the C terminus of Bur1 is not essential. Full-length Bur1 (BUR1) or the kinase domain lacking the C terminus (bur1-ΔC) complemented a Δbur1 strain, whereas the C terminus (bur1-C) did not. The BUR1 shuffle strain was transformed with pRS315-BUR1-TADH1, pRS315-bur1-ΔC-TADH1, pRS315-bur1-C-TADH1, and pRS315 and restreaked onto a 5-FOA-containing plate. C, the C terminus of Bur1 is sufficient for binding to RPA in vivo. RPA copurified with Bur1-C (amino acids 356–657) by TAP. D, mapping of the RID of Bur1. The binding of Bur1-C truncations to RPA was assessed in vitro. The indicated truncated versions of Bur1-C (amino acids are indicated on top of the gel) were expressed as GST fusion proteins in Escherichia coli, bound to GST beads, and incubated with RPA (purified from S. cerevisiae by TAP; lane 1; +) or buffer (−). GST served as negative control (lanes 2 and 3). The band corresponding to Rfa1 is indicated. The asterisk indicates a contamination from E. coli after purification on GSH beads. E, schematic of Bur1 domains sufficient for binding to RPA. For comparison, the CTD interaction domain (CID) as determined (5) is indicated. F, deletion of the C terminus of Bur1 causes sensitivity to transcription inhibitors and genotoxic agents. Cells expressing bur1-C had a minor growth defect on full medium (YPD) but exhibited pronounced sensitivity to drugs impairing transcription elongation (6-AU, 100 μg/ml), causing DNA damage (MMS, 0.035%), or causing replication stress (HU, 100 mm). The BUR1 shuffle strain was transformed with pRS315-BUR1-TADH1 and pRS315-bur1-ΔC-TADH1, pRS316-BUR1 was shuffled out on 5-FOA, and cells were spotted onto the indicated plates.

Synthetic genetic interaction between bur1-107 and recombination mutants Δrad52 and Δxrs2. A, BUR1 interacts genetically with RAD52. To test for a genetic interaction between BUR1 and RAD52, yeast cells with the indicated genotypes were tested for sensitivity to MMS by incubating 10-fold serial dilutions on plates containing various amounts of MMS (0.005% shown). BUR1 mutants showed synthetic sickness with deletion of RAD52. The BUR1 RAD52 double shuffle strain was transformed with pRS315-BUR1, pRS315-bur1-107, or pRS315-bur1-124 and pRS314-RAD52 or pRS314, and pRS316-BUR1 and pRS316-RAD52 were shuffled out using 5-FOA. B, BUR1 interacts genetically with XRS2. The wild type and single and double mutants of BUR1 and XRS2 with the indicated genotypes were tested for sensitivity to MMS as described for A. A bur1-107::LEU2 XRS2 shuffle strain was transformed with pRS315-BUR1 or pRS315 and pRS313-XRS2 or pRS313, and pRS316-XRS2 was shuffled out using 5-FOA. Plates were incubated for 2 days at 30 °C.

The bur1-107 mutation suppresses the HU sensitivity of a Δmec1 mutant but leads to synthetic sickness in the presence of MMS. 10-Fold serial dilutions of Δsml1, Δmec1 Δsml1 (Δmec1 is lethal in the presence of SML1), bur1-107 Δsml1, and Δmec1 bur1-107 Δsml1 strains were spotted onto plates containing the indicated amounts of MMS and HU and grown for 2 days at 30 °C.

Model for the role of Bur1-Bur2 in the DNA replication stress response. During S phase, stalled replication forks expose ssDNA, which is recognized by RPA. Stalled replication forks are transiently stabilized by the Mec1-dependent S phase checkpoint, which suppresses untimely initiation of homologous recombination at the replication fork. In the absence of MEC1, stalled replication forks collapse, and the Rad52 recombination protein is recruited as evidenced by the formation of Rad52 foci. Both the Rad52 and Mec1 pathways are controlled by RPA. Mutation of BUR1 leads to an increase in Rad52 foci during replication stress, suggesting that the Bur1-Bur2 complex contributes to stabilization of stalled replication forks. The suppression of Δmec1 HU sensitivity by bur1 mutation indicates that Bur1 directs stalled replication forks down the Mec1-dependent pathway. Furthermore, the synergistic MMS sensitivity of bur1 mutations with Δrad52, Δxrs2, and Δmec1 indicates that Bur1 also plays a role in directing repair of MMS-induced lesion by the base/nucleotide excision repair (BER/NER) pathways rather than by homologous recombination.

Transcription elongation factor Bur1-Bur2 interacts with RPA. A, purification of TAP-tagged versions of Bur1 and Bur2 copurifies RPA. EGTA eluates were separated by SDS gel electrophoresis and stained with Coomassie Blue. The RPA subunits Rfa1 and Rfa2 were identified as copurifiers by mass spectrometry (left panel). The interaction between Bur1-Bur2 and RPA depended on the presence of DNA. Whole cell extracts were treated with 100 μg/ml DNase for 30 min at 23 °C to eliminate DNA prior to purification (right panel). B, purification from a non-tagged wild-type, an Rfa1-TAP, and a Prt1-TAP strain. Whole cell extracts and EGTA eluates were separated by SDS gel electrophoresis and stained with Coomassie Blue. Copurification of Bur1 was specifically detected in the Rfa1-TAP purification by Western blotting. Black circles indicate TAP-tagged Rfa1 and Prt1, which were detected with the secondary antibody because of their protein A tag.

Mutation of BUR1 suppresses the DNA damage sensitivity of rfa1-249. A, sensitivity of bur1-ts mutants to drugs impairing transcription (6-AU, 100 μg/ml), causing DNA damage (MMS, 0.035%), or causing replication stress (HU, 100 mm). The BUR1 shuffle strain was transformed with pRS315-BUR1, pRS315-bur1-101, pRS315-bur1-104, pRS315-bur1-107, and pRS315-bur1-124, and pRS316-BUR1 was shuffled out using 5-FOA. B, mutation of BUR1 suppresses the growth impairment and sensitivity to MMS (0.005%) but slightly enhances the sensitivity to HU (25 mm) caused by mutation of RFA1. The BUR1 RFA1 double shuffle strain was transformed with pRS315-BUR1 or pRS315-bur1-107 and pRS314-RFA1 or pRS314-rfa1-249, and pRS316-BUR1 and pRS316-RFA1 were shuffled out using 5-FOA. C, bur1-ΔC is unable to revert the suppression of rfa1-249 by bur1-107 in contrast to wild-type BUR1. The BUR1 RFA1 double shuffle strain was transformed with pRS313-BUR1 and pRS314-RFA1 (first row); pRS313-bur1-107 and pRS314-RFA1 (second row); pRS313-BUR1 and pRS314-rfa1-249 (third row); pRS313-bur1-107 and pRS314-rfa1-249 (fourth row); pRS313-bur1-107, pRS314-rfa1-249, and pRS315-BUR1 (fifth row); pRS313-bur1-107, pRS314-rfa1-249, and pRS315-RFA1 (sixth row); and pRS313-bur1-107, pRS314-rfa1-249, and pRS315-bur1-ΔC (seventh row). pRS316-BUR1 and pRS316-RFA1 were shuffled out on 5-FOA, and cells were spotted onto SDC(−His/−Trp/−Leu) plates containing the indicated concentrations of MMS and HU. Plates were incubated for 2 days at 30 °C.

Increased formation of Rad52 foci in bur1 mutants. A, colocalizing Rfa1-RFP and Rad52-YFP foci in bur1 mutants. Wild-type and bur1-101, bur1-104, bur1-107, bur1-124, and bur1-ΔC mutant strains expressing Rfa1-RFP from the endogenous locus and Rad52-YFP ectopically (pWJ1213) (37) were grown in SDC(−His) at 30 °C. The occurrence of Rfa1-RFP and Rad52-YFP was determined by fluorescence microscopy before and after exposure to 100 mm HU for 1 h. Representative cells are shown for wild type and the bur1–107 mutant. Selected foci are indicated by arrowheads. DIC, differential interference contrast. B, quantitation of Rfa1 and Rad52 foci. For each genotype, 100–200 cells were inspected. Error bars indicate 95% confidence intervals. In the absence of HU, the percentage of cells with colocalizing Rfa1 and Rad52 foci was significantly higher compared with the wild type in the bur1-107, bur1-124, and bur1-ΔC mutants (p = 0.007, 1.63 × 10⁻⁸, and 2.59 × 10⁻⁸, respectively, by Fisher's exact test (one-tailed)). The bur1-101 and bur1-104 mutants displayed focus levels similar to those of the wild type (p = 0.26 and 0.48, respectively). C, Rad52 foci accumulate in HU-treated bur1-107 cells. The experiment was performed as described for B, except that cells were examined for Rad52 foci at 0, 1, 2, and 3 h after the addition of 100 mm HU. In untreated cells, the percentage of cells with spontaneous Rad52 foci was only slightly elevated over the wild type (p = 0.077). Upon treatment with HU for 1–3 h, the percentage of cells with Rad52 foci increased significantly over the wild type (p = 0.0007, 7.53 × 10⁻¹⁰, and 3.56 × 10⁻²², respectively, by Fisher's exact test (one-tailed)). D, Rad52 foci persist in a bur1-107 mutant. The duration of spontaneous Rad52 foci in the absence of exogenous DNA damage was determined for wild-type and bur1-107 cells by time-lapse microscopy for a period of 4–5 h. The percentage of cells that formed at least one Rad52 focus/cell cycle was 56% (67 of 119) for the wild type and 64% (49 of 77) for the bur1-107 mutant. The median duration of spontaneous Rad52 foci was significantly longer than the wild type for the bur1-107 mutant (p = 0.05, one-tailed Student's t test).

Transcriptome profiling analysis of bur1-107 and rfa1-249 mutants. A, histogram of genes exhibiting significantly altered mRNA levels in bur1-107, rfa1-249, and bur1-107 rfa1-249 cells after treatment with MMS (0.1%). The proportion of up- and down-regulated genes is indicated in red and green, respectively. The numbers of the respective genes are given. B, Venn diagram of the 115 differentially expressed genes. The corresponding numbers of genes are given within the circles. C, cluster analysis of the genome-wide expression profiles of bur1-107, rfa1-249, and the bur1-107 rfa1-249 double mutant. The cluster diagram was calculated for the corresponding 115 significantly altered genes. Both rows and columns were clustered using a hierarchical cluster algorithm (see “Experimental Procedures”). Rows represent individual genes, and -fold changes in gene expression are indicated by color intensity (intensity bar), with red, green, and black reflecting increase, decrease, and no change, respectively. Columns represent the different strains. The dendrogram for column clustering is shown. D, Pearson's correlation matrix for gene expression profiles of bur1-107, rfa1-249, and bur1-107 rfa1-249 strains. The corresponding correlation coefficients are given.