Role of Dot1 in the response to alkylating DNA damage in Saccharomyces cerevisiae: regulation of DNA damage tolerance by the error-prone polymerases Polzeta/Rev1

Abstract

Maintenance of genomic integrity relies on a proper response to DNA injuries integrated by the DNA damage checkpoint; histone modifications play an important role in this response. Dot1 methylates lysine 79 of histone H3. In Saccharomyces cerevisiae, Dot1 is required for the meiotic recombination checkpoint as well as for chromatin silencing and the G(1)/S and intra-S DNA damage checkpoints in vegetative cells. Here, we report the analysis of the function of Dot1 in the response to alkylating damage. Unexpectedly, deletion of DOT1 results in increased resistance to the alkylating agent methyl methanesulfonate (MMS). This phenotype is independent of the dot1 silencing defect and does not result from reduced levels of DNA damage. Deletion of DOT1 partially or totally suppresses the MMS sensitivity of various DNA repair mutants (rad52, rad54, yku80, rad1, rad14, apn1, rad5, rad30). However, the rev1 dot1 and rev3 dot1 mutants show enhanced MMS sensitivity and dot1 does not attenuate the MMS sensitivity of rad52 rev3 or rad52 rev1. In addition, Rev3-dependent MMS-induced mutagenesis is increased in dot1 cells. We propose that Dot1 inhibits translesion synthesis (TLS) by Polzeta/Rev1 and that the MMS resistance observed in the dot1 mutant results from the enhanced TLS activity.

Figure 1.—

Deletion of DOT1 increases the MMS sensitivity of the rad24, but not the rad9, checkpoint mutant. Tenfold serial dilutions of exponentially growing cells were spotted onto YPDA and 0.005% or 0.01% MMS plates. Strains are YP436 (wild type), YP520 (rad9), YP521 (rad24), YP522 (rad9 rad24), YP523 (rad9 dot1), YP524 (rad24 dot1), YP525 (rad9 rad24 dot1), and YP506 (dot1).

Figure 2.—

Deletion of DOT1 alleviates the MMS sensitivity of the yku80 and rad52 mutants. (A) Tenfold serial dilutions of exponentially growing cells were spotted onto YPDA plates incubated at 30° or 37° and a 0.02% MMS plate incubated at 30°. (B) Tenfold serial dilutions of exponentially growing cells were spotted onto YPDA and 0.005% MMS plates and incubated at 30° for 48 and 72 hr. Strains are BR1919α (wild type), YP513 (yku80), YP345 (dot1), YP514 (yku80 dot1), YP370 (rad52), YP371 (rad52 dot1), YP516 (rad52 yku80 dot1), and YP515 (rad52 yku80). (C) The dot1 mutant shows increased MMS resistance. Fivefold serial dilutions of exponentially growing cells were spotted onto YPDA and 0.01 or 0.02% MMS plates and incubated at 30° for 36, 48, and 60 hr. Strains are YP210 (wild type) and YP345 (dot1).

Figure 3.—

Suppression of MMS sensitivity of yku80 and rad52 by dot1 does not depend on Sir3. Tenfold serial dilutions of exponentially growing cells were spotted onto YPDA and a 0.03% MMS plate (A) or a 0.01% MMS plate (B). Strains are BR1919α (wild type), YP513 (yku80), YP514 (yku80 dot1), YP544 (sir3), YP547 (sir3 dot1), YP545 (yku80 sir3), YP546 (yku80 sir3 dot1), YP370 (rad52), YP515 (rad52 yku80), YP516 (rad52 yku80 dot1), YP548 (rad52 sir3), YP549 (rad52 sir3 dot1), YP550 (rad52 yku80 sir3), and YP551 (rad52 yku80 sir3 dot1).

Figure 4.—

Deletion of DOT1 does not suppress the sensitivity of the rad52 and yku80 mutants to a DSB generated by the HO endonuclease at the MAT locus (A) Fivefold serial dilutions of log-phase cells growing in YPDA were spotted onto YPDA or YP–galactose plates (bottom, left and middle, respectively). Fivefold serial dilutions of log-phase cells incubated in liquid YP–galactose for 4 hr were spotted onto a YPDA plate (bottom right). Strains are YP943 (wild type), YP944 (dot1), YP945 (rad52), and YP946 (rad52 dot1). (B) Viability of JKM179-derived strains lacking HMLα and HMRa after incubation in YP–galactose for 3 hr to induce HO expression. Strains are JKM179 (wild type), YP815 (dot1), YP1167 (yku80), and YP1168 (yku80 dot1). Average and standard deviation values of three independent experiments are shown.

Figure 5.—

Analysis of MMS-induced chromosome fragmentation by PFGE. (A) Exponentially growing wild-type (BR1919a) and dot1 (YP576) cells were treated with 0.05% MMS. After 15 and 30 min, agarose plugs were prepared and chromosomes were separated by PFGE. Chromosomes were visualized by ethidium bromide staining (left), transferred to a membrane, and hybridized with a probe specific for chromosome V (right). Chromosome V fragmentation results in a smear (bracket) below the band representing the whole chromosome (arrow). (B) Quantification of the radioactive signal present in A. Chromosome breakage is expressed as the ratio between the signal detected below the intact chromosome V band and the total signal detected on each lane.

Figure 6.—

MMS-induced Rad52 foci formation and histone H2AS129 phosphorylation are increased in the dot1 mutant. (A) Rad52-YFP foci in wild-type and dot1 cells treated with 0.02% MMS for 1 hr. Representative fields are shown. Images are the maximum intensity projection from z-stacks of eight sections separated by 0.4 μm. The percentage of cells containing Rad52 foci (B) and the number of Rad52 foci per cell (C) are represented. (D) Western blot analysis of histone H2A phosphorylation at serine 129 in wild-type and dot1 cells treated with 0.02% MMS. Phosphoglycerate kinase (PGK) was used as a loading control. Strains are W3749-14C (wild type) and YP741 (dot1).

Figure 7.—

Genetic interaction of dot1 with BER and NER mutants. (A) Deletion of DOT1 suppresses the MMS sensitivity of BER and NER mutants. Fivefold serial dilutions of exponentially growing cells were spotted onto YPDA and 0.01 or 0.015% MMS plates. Strains are BR1919α (wild type), YP163 (dot1), YP978 (rad1), YP981 (rad1 dot1), YP979 (rad14), YP987 (rad14 dot1), YP980 (apn1), and YP992 (apn1 dot1). (B) Suppression of the rad52 MMS sensitivity by dot1 does not depend on NER or BER function. Fivefold dilutions of exponentially growing cells were spotted onto YPDA and 0.001% MMS plates. Strains are BR1919α (wild type), YP370 (rad52), YP371 (rad52 dot1), YP983 (rad1 rad52), YP985 (rad1 rad52 dot1), YP989 (rad14 rad52), YP990 (rad14 rad52 dot1), YP994 (apn1 rad52), and YP995 (apn1 rad52 dot1).

Figure 8.—

Genetic interaction of dot1 with TLS mutants. (A) Deletion of DOT1 enhances the MMS sensitivity of the rev1 and rev3 mutants. Fivefold serial dilutions of exponentially growing cells were spotted onto YPDA and 0.005 or 0.01% MMS plates. Strains are BY4741 (wild type), BY4741-dot1Δ (dot1), BY4741-rad30Δ (rad30), YP1080 (rad30 dot1), BY4741-rev1Δ (rev1), YP1081 (rev1 dot1), BY4741-rev3Δ (rev3), and YP1082 (rev3 dot1). (B) Deletion of DOT1 partially suppresses the MMS sensitivity of the rad30 mutant. Fivefold dilutions of exponentially growing cells were spotted onto YPDA and 0.03% MMS plates. Strains are BY4741 (wild type), BY4741-dot1Δ (dot1), BY4741-rad30Δ (rad30), and YP1080 (rad30 dot1). (C) Suppression of the rad52 MMS sensitivity by dot1 requires Rev3 and Rev1 function. Fivefold dilutions of exponentially growing cells were spotted onto YPDA and 0.0005% MMS plates. Strains are BY4741 (wild type), YP811 (rad52), BY4741-rev3Δ (rev3), YP1125 (rad52 rev3), YP1126 (rad52 rev3 dot1), BY4741-rev1Δ (rev1), YP1196 (rad52 rev1), and YP1197 (rad52 rev1 dot1).

Figure 9.—

MMS-induced mutagenesis at the CAN1 locus is increased in the dot1 mutant. The mutagenesis frequency, expressed as the proportion of Can^R colonies that appeared in cultures from cells growing in the absence (A) or in the presence of 0.005% MMS (B), is represented. The average and standard deviation from four independent experiments is shown. Strains are BY4741 (wild type), BY4741-dot1Δ (dot1), BY4741-rev3Δ (rev3), and YP1082 (rev3 dot1).

Figure 10.—

Model for the role of Dot1 in the response to alkylating damage. MMS-induced damage can cause the stall of replication forks either directly or as a consequence of incomplete BER or NER. Stalled replication forks result in recombinogenic lesions, which can be repaired mainly by Rad52-dependent HR, with NHEJ acting as a minor repair pathway for DSBs. Replication blocks can also be bypassed by the action of TLS pathways. Dot1 functions as a negative regulator of Polζ- and Rev1-dependent TLS and is also required for efficient HR. In the absence of Dot1, the enhanced TLS activity results in fewer stalled replication forks, leading to increased MMS resistance.