International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int. J. Mol. at multiple different origins throughout the genome, but in contrast to S. cerevisiae where Sci. 2021, 22, 9409. https://doi.org/ origins are characterized by a specific sequence, origins in plants appear to be rather 10.3390/ijms22179409 specified by a certain epigenetic state [1]. Origin firing, the process of activating an origin, is strictly controlled by cyclin-dependent kinases (CDKs) in complex with their regulatory Academic Editor: Jean Molinier cyclin subunits. A plethora of proteins are involved in the process of DNA replication, including PCNA, MCMs, DNA polymerase α, δ, and ", chromatin assembly factors, and Received: 8 July 2021 others (for a recent review, see [2]). Among the polymerases, DNA polymerase α is a Accepted: 26 August 2021 protein complex consisting of a catalytic subunit, an accessory subunit, and two RNA Published: 30 August 2021 primases. The primase subunits are responsible for initiating replication at both leading and lagging strands by synthesizing around 10 nucleotides of RNA primer. After this, Publisher’s Note: MDPI stays neutral the primase subunit is displaced by the catalytic polymerase subunit and another 20–30 with regard to jurisdictional claims in deoxyribonucleotides are synthesized [3,4]. However, the DNA polymerase α processivity published maps and institutional affil- is very low and its exonuclease domain is inactive, causing it to have no proofreading iations. capability. Until recently, it was thought that DNA polymerase α is replaced rather quickly by DNA polymerase " on the leading strand and DNA polymerase δ on the lagging strand [5]. Recent work, however, suggests that DNA polymerase δ synthesizes both strands, whereas DNA polymerase " is rather involved in removing replication errors of Copyright: © 2021 by the authors. polymerase δ and playing a scaffolding role at the replication fork [6]. Licensee MDPI, Basel, Switzerland. Next to their role in DNA synthesis, over the years it has become increasingly clear This article is an open access article that polymerases play a role in epigenetic inheritance in plants. In genetic screens looking distributed under the terms and for repressors of DNA methylation-dependent gene silencing, different mutant alleles of conditions of the Creative Commons all three polymerases have been identified [7–10]. Two reported mutants in polymerase Attribution (CC BY) license (https:// α, called incurvata2-1 and pola-1, display similar phenotypes, being smaller leaves and creativecommons.org/licenses/by/ 4.0/). early flowering [8,11], which were shown to originate from loss of epigenetic repression of Int. J. Mol. Sci. 2021, 22, 9409. https://doi.org/10.3390/ijms22179409 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9409 2 of 12 flowering-specific genes. Polymerase α interacts with TFL2/LHP1, a protein responsible for depositing heterochromatin marks on histones, and in this way, it could help to maintain repressive histone marks at the replication fork. Similarly, missense mutants of polymerases δ and " display, next to increased sensitivity to certain kinds of DNA damage, accelerated flowering that is correlated with changes in heterochromatin marks such as H3K27me3 and H3K4me3 [7,9,12,13]. This is similar to the situation in fission yeast, where the catalytic subunit of polymerase α binds to the LHP1 homolog in order to maintain heterochromatin structure by recruiting histone methyltransferases [14,15]. To safeguard genomic integrity and allow faithful transmission of the DNA to their progeny, eukaryotes have developed extensive mechanisms and signaling pathways that control the fidelity of the DNA replication process. In yeast and mammals, where this process has been widely studied due to its link with oncogenesis, there are two ser- ine/threonine protein kinases called ATAXIA TELANGIECTASIA MUTATED (ATM) and ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (ATR) that are involved in the sensing and responding to double-stranded breaks (DSBs) and single-stranded DNA (ssDNA), respectively. Downstream of these two sensor kinases two checkpoint kinases, Chk1 and Chk2, act upon a plethora of downstream targets, including the p53 transcription factor that induces expression of DNA repair genes, cell cycle regulators, and possible cell death-inducing genes [16]. In plants, the upstream ATM and ATR kinases are conserved, but the checkpoint kinases Chk1 and Chk2 and the transcription factor p53 are missing, although plants have a functional homolog called SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) [17]. During replication, the most common type of damage that occurs is a stalled replica- tion fork, which can arise at any point where the progress of the DNA processing enzymes is blocked. Endogenous causes include bulky nucleotides (e.g., through alkylation), short- age of dNTPs, crosslinked DNA, protein–DNA adducts, etc. Exogenous causes of stalled replication forks are mostly pyrimidine dimers caused by UV radiation. A stalled repli- cation fork leads to a stretch of single-stranded DNA (ssDNA) being unwound by the uncoupling of the helicase from the replisome, resulting in activation of ATR [18]. This activates a signaling cascade called the intra-S replication checkpoint that, amongst others, causes stabilization of the stalled replication forks [19]. Next to stabilizing existing replica- tion forks, the replication checkpoint also slows down the firing of new origins, a process in humans that is dependent on WEE1-mediated CDK inhibition [20]. WEE1 is a tyrosine kinase, which phosphorylates CDKs to inhibit their activity. In the absence of WEE1, new replication forks continue to be initiated and will then stall and collapse, leading to double-strand breaks (DSBs) [20]. In Arabidopsis, both ATR and WEE1 are necessary to survive replication stress [21,22], with mutants showing drastically shortened roots when grown on medium containing chemicals that induce replication stress, such as hydroxyurea (HU). Here, we report the identification of a suppressor mutation in the gene encoding the catalytic subunit of Arabidopsis DNA polymerase α able to partially rescue root growth of wee1 plants on HU-containing medium. We furthermore show that this mutation has an effect on the duration of the DNA replication phase, likely by limiting the availability of functional DNA polymerase α complexes. 2. Results 2.1. A Mutation in the Catalytic Subunit of Polymerase a Rescues HU Sensitivity of WEE1KO Plants When WEE1KO plants are grown on a medium containing HU, they show strong inhibition of root growth [22]. An ethyl methanesulfonate (EMS) mutagenesis screen was performed to identify mutations that can rescue this root growth inhibition. One of the revertants found was line 113–2, which showed improved root growth on HU compared to wee1-1 plants. By outcrossing this line to a wee1-1 mutant line in Landsberg erecta background, a mapping population was obtained that could be used for next-generation sequencing-based mutation mapping using the SHORE pipeline coupled to the SHOREmap Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12 Int. J. Mol. Sci. 2021, 22, 9409 3 of 12 background, a mapping population was obtained that could be used for next-generation sequencing-based mutation mapping using the SHORE pipeline coupled to the SHORE- algorithmmap algorithm [23]. The[23]. causalThe causal mutation mutation of line of 113–2line 113–2 was was mapped mapped to a to genomic a genomic region region on theon the lower lower arm arm of chromosomeof chromosome 5, more5, more exactly exactly to to the theAt5G67100 At5G67100gene, gene, where where it it leads leads to to thethe nonsynonymousnonsynonymous P846SP846S aminoamino acidacid changechange (Figure(Figure1 A).1A). The The mutation mutation was was confirmed confirmed inin thethe originaloriginal 113–2113–2 lineline byby direct direct Sanger Sanger sequencing sequencing (Figure(Figure S1).S1).