Analysis of the tolerance to DNA alkylating damage in MEC1 and RAD53 checkpoint mutants of Saccharomyces cerevisiae

Abstract

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.

Figure 1. Analysis of DNA damage tolerance pathway in rad53Ha mutants.

(A) Mutation of endonuclease SLX4 or DNA repair protein ESC4/RTT107 has little impact on MMS sensitivity of rad53Ha cells. Serial dilutions (ten-fold) of wild-type, slx4Δ, esc4Δ, rad53Ha, slx4Δ rad53Ha and esc4Δ rad53Ha cells were plated onto YPAD plates with and without MMS (as indicated). (B) Analysis of the effect of MMS2 mutation on rad53Ha cells tolerance. As in (A) ten-fold serial dilutions of the indicated strains were plated onto YPAD plates with and without MMS. (C) Deletion of REV1, REV3 or REV7 suppresses resistance to MMS in rad53Ha cells. Ten-fold serial dilutions of the indicated strains are shown. (D) Relative balance of the error-prone and error-free tolerance pathways in rad53Ha cells. Serial dilutions (ten-fold) of wild-type, rad53Ha, mms2Δ, rev3Δ, mms2Δ rad53Ha and rev3Δ rad53Ha plated onto YPAD plates with 0.005% MMS or without the alkylating chemical. YPAD plates (YPD supplemented with 50 µg/ml Adenine) containing MMS were freshly made and used within 5–7 hours. Note: All the experiments shown in this work were repeated three times and with different clones of every mutant to ensure reproducibility.

Figure 2. The Lysine 164 to Arginine mutation of POL30 suppresses the MMS resistance of rad53Ha.

(A) Serial dilutions (ten-fold) of wild-type, rad53Ha, pol30^K164R and pol30^K164R rad53Ha cells were plated onto YPAD plates with and without MMS (as indicated). (B) Deletion of MMS2 and REV3 prevents tolerance to MMS in rad53Ha cells. Ten-fold serial dilutions on YPAD plates with or without MMS of the indicated strains are shown. (C) Resistance to MMS in rad53Ha cells is independent from PCNA SUMOylation. Ten-fold serial dilutions of wild-type, rad53Ha, siz1Δ and siz1Δ rad53Ha were plated onto YPAD plates with and without MMS. (D) Mutation of RAD18 E3 ubiquitin ligase prevents tolerance to MMS-mediated DNA damage in rad53Ha mutants. Serial dilutions (ten-fold) of wild-type, rad18Δ and rad18Δ rad53Ha cells were plated on YPAD plates with and without MMS.

Figure 3. Analysis of the DNA damage tolerance pathway in mec1 Δ and rad53 Δ checkpoint kinase mutants.

Serial dilutions (ten-fold) of indicated strains (see below) plated on YPAD plates with MMS and without the alkylating chemical. (A) wild-type, rad53Δ sml1Δ, mms2Δ, rev3Δ, mms2Δ rad53Δ sml1Δ and rev3Δ rad53Δ sml1Δ. (B) wild-type, mec1Δ sml1Δ, mms2Δ, rev3Δ, mms2Δ mec1Δ sml1Δ and rev3Δ mec1Δ sml1Δ. Viable plot graphs in (A) and (B) show the viability analysis of wild-type, rad53Δ sml1Δ and mec1Δ sml1Δ strains in mms2Δ or rev3Δ backgrounds (as indicated).

Figure 4. Phosphorylation of Mec1 and Mec1/Tel1 substrates in rad53 mutant cells.

TCA-extracted protein samples were taken from untreated cells (U) or 90 minutes MMS-treated cells (0.02% MMS except where indicated) (M), processed for Western blotting after SDS-PAGE in appropriate gels (from 8% to 12%), and probed with α-γH2AX (ab15083, Abcam), α-myc (M^.5546, Sigma-Aldrich), α-Ha (12CA5, Sigma-Aldrich) or α-Rad53 (sc-6749, Santa Cruz Biotechnology Inc.) antibodies to detect histone H2A phosphorylated at S129, myc-tagged Slx4, Ha-tagged Ddc1 and Rad53, respectively. (A) γH2AX antibody immunoreactivity of whole cell extracts from wild-type and rad53Ha (r53Ha), rad53^S350A,G404V (r53^350,404) and rad53Δ (r53Δ) mutants untreated (U) or MMS-treated (M) as indicated. (B) (Upper panel) γH2AX antibody immunoreactivity and (middle panel) myc antibody immunoreactivity of whole cell extracts from slx4-myc-tagged cells, otherwise wild-type (wt), rad53Ha (r53Ha), rad53Δ (r53Δ) or mec1Δ, untreated (U) or MMS-treated (M). (C) Detection of γH2A (upper blot), Ddc1-Ha and Rad53 (lower blots) in whole cell extracts from ddc1-Ha-tagged cells, otherwise wild-type (wt), mec1-1, rad53Ha (r53Ha) or rad53Δ (r53Δ), untreated (U) or MMS-treated (M).

Figure 5. Analysis of the DNA damage tolerance pathway in mec1 Δ rad53 Δ double mutants.

Serial dilutions (ten-fold) of indicated strains (see below) plated on YPAD plates with MMS or without the alkylating chemical. (A) wild-type, mec1Δ sml1Δ, mms2Δ, rev3Δ, mec1Δ mms2Δ sml1Δ, mec1Δ rev3Δ sml1Δ, rad53Δ sml1Δ, rad53Δ rev3Δ sml1Δ, rad53Δ mms2Δ sml1Δ, mec1Δ rad53Δ sml1Δ, mec1Δ rad53Δ mms2Δ sml1Δ and mec1Δ rad53Δ rev3Δ sml1Δ. (B) Viability analysis of wild-type and mec1Δ rad53Δ sml1Δ strains in mms2Δ or rev3Δ backgrounds (as indicated).

Figure 6. Model for Mec1p and Rad53p regulatory network in the tolerance pathway.

In S.cerevisiae, a complex network links the S-phase checkpoint and the DNA damage tolerance pathway. We suggest that the opposite effects of two key S-phase checkpoint in yeast -the sensor kinase Mec1 and the effector kinase Rad53- help to maintain the balance of the two branches of the DNA damage-tolerance pathway. We propose that Rad53 would inhibit TLS DNA polymerase activity at least by limiting Rev1 foci formation. We also suggest that Mec1 would counterbalance Rad53 action on TLS polymerases, activating them either directly or through one of their positive effectors. At the same time Mec1 would also modulate Rad53 inhibitory effect through Rad53 activation. Additionally, Mec1 could activate the (error-free) template-switching branch of the tolerance pathway.