The Oxidative Stress Responsive Transcription Factor Pap1 Confers DNA Damage Resistance on Checkpoint-Deficient Fission Yeast Cells

<div><p>Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to <i>chk1</i>-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of <i>chk1</i>-deficient cells with mutations in genes encoding DNA ligase (<i>cdc17</i>) or DNA polymerase δ (<i>cdc6</i>), but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to <i>chk1</i> mutants, as it also improved survival of <i>rad1</i>- and <i>rad9</i>-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active.</p></div

Pap1 does not rescue mitotic catastrophe due to loss of Cdc2 tyrosine 15 phosphorylation or UV sensitivity of <i>chk1</i>-deficient cells.

<p>(A) Transformants of a <i>wee1–50 chk1Δ</i> double mutant strain carrying pSP1, pChk1, or pPap1 were spotted onto YEA agar medium and incubated at the permissive temperature of 25°C or restrictive temperature of 36°C for 3–5 days. (B) <i>WT, chk1Δ, and chk1D469G</i> cells without or with Pap1 overexpression were plated on YEA agar medium and exposed to 0, 40, 80, and 120 J/m² of UV light. Plates were incubated at 30°C for 3 to 5 days until colonies formed.</p

Pap1 rescues CPT sensitivity of other DNA damage checkpoint pathway mutants.

<p>(A) <i>rad3-T80 chk1D469G</i> strains carrying pSP1, pChk1, or pPap1 plasmids were spotted onto YEA medium in the absence or presence of 1 µM or 3 µM CPT. Plates were incubated at 30°C for 3–5 days. (B) <i>WT</i> cells, as well as <i>rad1Δ</i> and <i>rad9Δ</i> cells with or without pPap1 expression were spotted onto YEA medium in the absence or presence of 10 µM or 15 µM CPT.</p

Pap1 improves survival of <i>chk1</i>-deficient cells with particular DNA replication defects.

<p>(A) A <i>chk1</i>-deficient strain with temperature sensitive DNA ligase, <i>cdc17-k42 chk1Δ</i> was transformed with an empty vector (pSP1), pChk1 or pPap1. Cells were grown overnight at 25°C, then serially diluted and spotted onto PMA plates at 25°C and the restrictive temperature for the double mutant of 32°C. (B) A <i>chk1</i>-deficient strain with temperature sensitive DNA polymerase δ, <i>cdc6–23 chk1Δ</i>, was transformed with an empty vector (pSP1), pChk1 or pPap1. Cells were grown overnight at 25°C, then serially diluted and spotted onto PMA plates at the restrictive temperature of 29°C. (C) <i>Chk1</i>-deficient strains with temperature sensitive alleles of an MCM subunit (<i>cdc21-M68 chk1Δ</i>) or the MCM complex loader (<i>cdc18-K46 chk1Δ</i>) were transformed with an empty vector (pSP1), pChk1 or pPap1. Cells were grown overnight at 25°C, then serially diluted and spotted onto PMA plates at temperatures restrictive for the double mutants, 30°C for <i>cdc21-M68 chk1Δ</i> and 32°C for <i>cdc18-K46 chk1Δ</i>.</p

Pap1 does not appear to accumulate in the nucleus in response to CPT.

<p><i>WT, chk1Δ, and chk1D469G</i> strains expressing GFP-tagged Pap1 under control of the <i>nmt41</i> promoter were exposed to 40 µM CPT at 30°C. Cells were collected after 30 minutes, 2 hours and 3 hours for analysis by fluorescence microscopy.</p

Chk1 does not affect Pap1 localization in response to H₂O₂.

<p><i>WT, chk1Δ, and chk1D469G</i> strains expressing GFP-tagged Pap1 under control of the <i>nmt41</i> promoter were treated with 0.9 mM H₂O₂ for 15 minutes followed by analysis by fluorescence microscopy. Cells in the right column were treated with 0.9 mM H₂O₂ for 15 minutes and washed with fresh media to remove the drug before analysis.</p

Nuclear Pap1 improves growth of checkpoint-deficient cells with defective DNA ligase or DNA polymerase δ.

<p>(A,B) Checkpoint and DNA ligase-deficient <i>cdc17-K42 chk1D469G</i> double mutants (A) or checkpoint and DNA polymerase δ-deficient <i>cdc6–23 chk1D469G</i> double mutants (B) expressing the Pap1 constructs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089936#pone-0089936-g007" target="_blank">figure 7A</a> were spotted onto PMA medium and incubated at 25° and 32°C.</p

(A) Behavior of sister centromeres (white and red arrowheads) and the SPB (yellow) at MII in (top left and right) and (bottom left and right) mutants

The graph shows changes in the SPB-cen (D1 and D2) and SPB-SPB (D3) distances. PI, phase I; PII, phase II; PIII, phase III. Bar, 2 μm. (B) Mean distances of centromeres at phase II. More than eight pairs of centromeres were examined for each analysis. (C) Changes in spindle length at MII in the (left) and (right) mutants. Graphs show kinetics of two MII spindles in the same cell. Dotted lines in graphs show boundaries of the spindle phases. (D) Duration of the spindle phases at MII. (E) Duration of MI, MII, and the MI–MII interval. Wt, wild type. Error bars indicate standard deviation.<p><b>Copyright information:</b></p><p>Taken from "Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation"</p><p></p><p>The Journal of Cell Biology 2008;182(2):277-288.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483520.</p><p></p