Role of Dot1 in the Response to Alkylating DNA Damage in Saccharomyces cerevisiae: Regulation of DNA Damage Tolerance by the Error-Prone Polymerases Polζ/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 Polz/Rev1 and that the MMS resistance observed in the dot1 mutant results from the enhanced TLS activity.

Interplay Between Histone H3 Lysine 56 Deacetylation and Chromatin Modifiers in Response to DNA Damage

“…Our results also argue that Rad53-mediated inhibition of translesion DNA synthesis (TLS) contributes to the MMS sensitivity of hst3D hst4D mutants (Figure 6,H and I). These data are in line with published results showing that reduction of DDR kinase activity and concomitant increase in TLS partially suppress the MMS sensitivity caused by deletion of genes such as RAD52 or RTT107 (Conde and San-Segundo 2008;Conde et al 2010;Lévesque et al 2010). Interestingly, deletion of the REV3 gene encoding the catalytic subunit of translesion DNA polymerase zeta does not influence suppression of the Ts-phenotype of hst3D hst4D cells by dot1D, H3K79R, or H4K16R (Figure 6, H and I).…”

“…Previously published data indicate that cells lacking Dot1 are more resistant to MMS-induced DNA damage than WT cells, and this increased resistance to MMS has been correlated with reduced levels of Rad53 activation (Conde and San-Segundo 2008;Conde et al 2010;Lévesque et al 2010). The sensitivity to MMS of certain yeast mutants (including rad52D and rtt107D) is also reduced in the absence of Dot1 or H3K79 trimethylation (Conde and San-Segundo 2008;Conde et al 2010;Lévesque et al 2010).…”

“…The sensitivity to MMS of certain yeast mutants (including rad52D and rtt107D) is also reduced in the absence of Dot1 or H3K79 trimethylation (Conde and San-Segundo 2008;Conde et al 2010;Lévesque et al 2010). We sought to verify whether the sensitivity to MMS of mutants of the H3K56Ac pathway also was suppressed by DOT1 mutations.…”

The Ddc2/ATRIP checkpoint protein monitors meiotic recombination intermediates

“…TCA cell extracts from 5-10 ml aliquots of meiotic cultures were prepared and analyzed as described (Conde and San-Segundo, 2008). In-house produced mouse monoclonal anti-HA and rabbit polyclonal anti-GFP were used at 1:2000 dilution.…”

“…Dityrosine fluorescence (San-Segundo and Roeder, 2000) and DNA damage sensitivity assays (Conde and San-Segundo, 2008) have been previously described. To calculate the statistical significance of differences, a two-tailed Student t-test was used.…”