Identification and characterization of mutants with increased DNA stress resistance in Arabidopsis thaliana and Zea mays

01 January 2014 → 31 December 2017
Regional and community funding: IWT/VLAIO
Research disciplines
  • Natural sciences
    • Plant biology
zea mays Arabidopsis thaliana cell cycle regulation sessile organisms
Project description

Plants are sessile organisms, meaning that in contrast to mammals they cannot escape adverse growth conditions such as drought, heat, excess UV radiation, etc. For this reason, they have evolved extensive mechanisms to safeguard their genomic integrity, in order to pass on an intact copy of their genome to the offspring. During this PhD, we aimed to learn more about cell cycle regulation, and more specifically about the molecular mechanism behind DNA damage-induced checkpoints. We were interested in characterizing of the e2fa e2fb double mutant (hereafter called e2fab), mutated in both G1/S transcriptional activators. We found that the mutant has less cells in the root meristem and in mature leaves, and this is correlated with an increase both in S-phase and in total cell cycle duration, indicating checkpoint activation at possibly several different cell cycle phases. Surprisingly, no differential growth was seen under several different DNA damage treatments. A transcriptome study of this mutant revealed that next to downregulated E2F-dependent genes, a whole cluster of genes involved in the DNA damage response (DDR) was upregulated. Because of the link with transcriptional activation of the DDR, we crossed the e2fab mutant to the sog1 mutant and found that this triple mutant was hypersensitive to replication stress, indicating that E2Fs play a role next to SOG1 in the response to replication stress. Next to this, we identified and characterized three suppressor mutants of the wee1 mutant, which is hypersensitive to DNA replication stress. This results in severely reduced root growth when these plants are grown on medium containing hydroxyurea (HU), a drug that causes replication stress by lowering the available dNTP pools. The suppressor mutants can all partially rescue this hypersensitive phenotype of wee1 on HU, and they do this through different mechanisms. The trd2-1 mutant is mutated in subunit B of the ribonuclease H2 (RNase H2) complex, and shows the same characteristics as the previously identified trd1-1 mutant, mutated in subunit A of this complex. These mutants accumulate rNTPs into the genome, in this way probably compensating for the lack of available dNTPs caused by HU. The second identified suppressor mutant is a knockout of the FAS1 gene, mutated in a subunit of the chromatin assembly factor 1 (CAF-1) complex that is responsible for the correct formation of nucleosomes after DNA replication by adding histones H3 and H4 to the newly synthesized DNA strand. Absence of functional CAF-1 leads to activation of an ATM- and SOG1- dependent G2/M-checkpoint, in this way making the ATR- and WEE1-mediated intra-S checkpoint obsolete. Furthermore, we found that other phenotypes of the fas1 mutant, such as telomere shortening and loss of ribosomal DNA copies, are caused by the activated ATM-pathway, as evidenced by a (partial) rescue of telomere length and ribosomal DNA copy number in the fas1 atm double mutant. Lastly, we identified the missense pol-2 mutation, mutated in the catalytic subunit of DNA polymerase alpha, that rescues wee1 hypersensitivity to HU through an as yet not fully characterized mechanism. Preliminary results indicate that the mutated form is less stable, resulting in a loss of interaction with the primase subunits and possibly leading to a reduction in the amount of fired origins and thus a slowdown of replication phase. This theory is corroborated by the longer duration of Sphase in these plants. While research in model species such as Arabidopsis is important to identify new players in the DNA damage pathways, the genome of this small plant is a poor substitute for the much larger genome of crop species such as maize. To study and better understand the molecular response of maize to DNA damage, we generated knockout lines of all major DDR signalling players by using the CRISPR/Cas9 technique. Although characterization of some of these knockout lines fell outside the scope of this PhD project, we could nevertheless show that the knockout lines of ATR and WEE1 share the phenotypes that were already described in the corresponding Arabidopsis mutants. Furthermore, although maize ATR mutants are indistinguishable from the isogenic WT line in greenhouse conditions, they show a growth penalty when grown in the field, indicating that field conditions trigger DNA damage that requires ATR to ensure optimal growth. As a last project, we also identified and characterized the maize family of SIAMESE-RELATED CDK inhibitors, focussing on members that responded to DNA damage. In this way, we generated reporter lines and knockout lines of two members of this family which were then studied under different conditions. In summary, by studying the DNA damage response and its regulation of the cell cycle in both Arabidopsis and maize, we have gained a better understanding of how these two processes interact. In addition, we found a new role for E2Fs in the response to replication stress. Lastly, we generated a set of maize mutants that can be used to further study the DDR in an important crop species.