Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress

DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high‐throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin‐treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C‐terminal dual serine target motif unique to H2AX in the plant lineage showed 171‐fold phosphorylation that was absent in atm mutant lines. The physiological significance of post‐translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM‐mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post‐translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.     

Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.     

The interplay of DNA polymerase λ in diverse DNA damage repair pathways in higher plant genome in response to environmental and genotoxic stress factors

DNA repair mechanisms are essential for the maintenance of genomic stability, proper cellular function and survival for all organisms. Plants, with their intrinsic immobility, are vastly exposed to a wide range of environmental agents and also endogenous processes which frequently cause damage to DNA and impose genotoxic stress. Therefore, in order to survive under frequent and extreme environmental stress conditions, plants have developed a vast array of efficient and powerful DNA damage repair mechanisms to ensure rapid and precise repair of genetic material for maintaining genome stability and faithful transfer of genetic information over generations.¹ Recently, we have defined the role of DNA polymerase λ in repair of UV-B-induced photoproducts in Arabidopsis thaliana via nucleotide excision repair pathway.² Here, we have further discussed potential function of DNA polymerase λ in various DNA repair pathways in higher plant genome in response to environmental and genotoxic stress factors.     

ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background

The ataxia-telangiectasia mutated and rad3-related (ATR) kinase orchestrates cellular responses to DNA damage and replication stress. Complete loss of ATR function leads to chromosomal instability and cell death. However, heterozygous ATR mutations are found in human cancers with microsatellite instability, suggesting that ATR haploinsufficiency contributes to tumorigenesis. To test this possibility, we generated human cell line and mouse model systems in which a single ATR allele was inactivated on a mismatch repair (MMR)-deficient background. Monoallelic ATR gene targeting in MLH1-deficient HCT 116 colon carcinoma cells resulted in hypersensitivity to genotoxic stress accompanied by dramatic increases in fragile site instability, and chromosomal amplifications and rearrangements. The ATR(+/-) HCT 116 cells also displayed compromised activation of Chk1, an important downstream target for ATR. In complementary studies, we demonstrated that mice bearing the same Atr(+/-)/Mlh1(-/-) genotype were highly prone to both embryonic lethality and early tumor development. These results demonstrate that MMR proteins and ATR functionally interact during the cellular response to genotoxic stress, and that ATR serves as a haploinsufficient tumor suppressor in MMR-deficient cells.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

A positive role for c-Abl in Atm and Atr activation in DNA damage response

DNA damage triggers Atm- and/or Atr-dependent signaling pathways to control cell cycle progression, apoptosis, and DNA repair. However, how Atm and Atr are activated is not fully understood. One of the downstream targets of Atm is non-receptor tyrosine kinase c-Abl, which is phosphorylated and activated by Atm. The current view is that c-Abl relays pro-apoptotic signals from Atm to p73 and p53. Here we show that c-Abl deficiency resulted in a broad spectrum of defects in cell response to genotoxic stress, including activation of Chk1 and Chk2, activation of p53, nuclear foci formation, apoptosis, and DNA repair, suggesting that c-Abl might also act upstream of the DNA damage-activated signaling cascades in addition to its role in p73 and p53 regulation. Indeed, we found that c-Abl is required for proper activation of both Atm and Atr. c-Abl is bound to the chromatin and shows enhanced interaction with Atm and Atr in response to DNA damage. c-Abl can phosphorylate Atr on Y291 and Y310 and this phosphorylation appears to have a positive role in Atr activation under genotoxic stress. These findings suggest that Atm-mediated c-Abl activation in cell response to double-stranded DNA breaks might facilitate the activation of both Atm and Atr to regulate their downstream cellular events.     

ATM/ATR-dependent Tyr15 phosphorylation of cyclin-dependent kinases in response to hydroxyurea in Vicia faba root meristem cells

DNA damage or stalled replication forks activate cell cycle checkpoints. However, the regulation of metabolic pathways that are responsible for maintenance of genome integrity in plants is still largely unknown. Present research on Vicia faba root meristem cells indicates that inhibitory phosphorylation of cyclin-dependent kinases (Cdks) at Tyr15 plays a prominent role during blockage of cell cycle in response to genotoxic stress. Phosphorylation of P-loop in Cdks takes place in ATM/ATR-dependent manner. Although, Tyr15 phosphorylation upon hydroxyurea (HU) treatment was found in most cells classified to G1 and S phase, interestingly, the number of phoshpo-Tyr15-positive cells decreases in G2 phase. Presented data confirm much similarity in regulation of Cdks functions under genotoxic stress between plants and animals; however, they may also substantiate evolutionarily developed differences especially in regulation of G2/M transition between these two kingdoms.     

Loss of mitochondrial DNA under genotoxic stress conditions in the absence of the yeast DNA helicase Pif1p occurs independently of the DNA helicase Rrm3p

How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal conditions and in the presence of genotoxic stress is less understood. We demonstrate that the inefficient mtDNA replication process of mutant yeast cells lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA helicase RRM3. The rescue effect is likely due to the increase in the deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of genotoxic stress is not suppressed if RRM3 is lacking suggesting that this phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found to stimulate the incorporation of dNTPs into newly synthesised mtDNA of gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA helicase in mitochondria affects mtDNA replication directly. Possible roles of Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the temporarily displacement of tightly bound mtDNA-binding proteins, or the stabilization of the mitochondrial replication complex during mtDNA replication. X. Cheng, Y. Qin contributed equally to this work.     

Autophagy and genomic integrity

DNA lesions, constantly produced by endogenous and exogenous sources, activate the DNA damage response (DDR), which involves detection, signaling and repair of the damage. Autophagy, a lysosome-dependent degradation pathway that is activated by stressful situations such as starvation and oxidative stress, regulates cell fate after DNA damage and also has a pivotal role in the maintenance of nuclear and mitochondrial genomic integrity. Here, we review important evidence regarding the role played by autophagy in preventing genomic instability and tumorigenesis, as well as in micronuclei degradation. Several pathways governing autophagy activation after DNA injury and the influence of autophagy upon the processing of genomic lesions are also discussed herein. In this line, the mechanisms by which several proteins participate in both DDR and autophagy, and the importance of this crosstalk in cancer and neurodegeneration will be presented in an integrated fashion. At last, we present a hypothetical model of the role played by autophagy in dictating cell fate after genotoxic stress.     

Genotoxic stress-activated DNA-PK-p53 cascade and autophagy cooperatively induce ciliogenesis to maintain the DNA damage response

The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.     

Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.     

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.     

Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype,enhances genome stability and changes chromatin compaction

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.