Ribonucleotide Reductase Regulation in Response to Genotoxic Stress in Arabidopsis

Abstract

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

“…In Arabidopsis, bleomycin triggers a DSB stress response (Menke et al, 2001;Tamura et al, 2002;Chen et al, 2008), whereas hydroxyurea induces a replication stress response (Ferreira et al, 1994;Roa et al, 2009). Treating wild-type and Whirly mutant plants with bleomycin (see Supplemental Figure 7 online) or hydroxyurea (see Supplemental Figure 8 online) did not result in an increase in plastid or mitochondrial MHMR.…”

“…Bleomycin and hydroxyurea treatments were performed as described (Chen et al, 2008;Roa et al, 2009). The why2-1 and why1 why3 mutant lines (formerly called KO2 and KO1/3, respectively) were described previously (Maré chal et al, 2008(Maré chal et al, , 2009).…”

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis

“…Since DNA damage highly accumulated in the ae7 mutant, we next examined expression of several genes that are often induced in the DNA damage response. The tested genes included PARP genes that encode poly (ADP-ribose) polymerases required for the maintenance of DNA integrity during replication (DoucetChabeaud et al, 2001), BREAST CANCER SUSCEPTIBILITY1 (BRCA1) and GAMMA RESPONSE1 (GR1) that are involved in DSB repair by the homologous recombination (HR) pathway (Lafarge and Montané, 2003;Bleuyard et al, 2005), KU70, KU80, and LIGASE4 (LIG4) that are required for DSB repair by the nonhomologous end joining pathway (Tamura et al, 2002;van Attikum et al, 2003), TSO2 that is activated in response to DSBs, and RIBONUCLEOTIDE REDUCTASE 2A (RNR2A) and RNR2B that are specifically induced by hydroxyurea (Wang and Liu, 2006;Roa et al, 2009). Our real-time quantitative RT-PCR (qRT-PCR) showed that among the genes tested, transcript levels of PARP1, PARP2, BRCA1, GR1, and TSO2 were elevated in ae7 (Figure 2A).…”

The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans

“…HU induces replication stress by inhibiting the RIBONUCLEOTIDE REDUCTASE (RNR) required for dNTP synthesis. 30 This reversibly blocks the replication fork in S-phase and may also cause DNA fragmentation and chromosomal abnormalities. 30,31 If the putative Arabidopsis CDC25 gene has the conserved Cdc25 function, we expected that overexpressing plants should show hypersensitivity towards genotoxic stress similar to the situation in human cells.…”

“…30 This reversibly blocks the replication fork in S-phase and may also cause DNA fragmentation and chromosomal abnormalities. 30,31 If the putative Arabidopsis CDC25 gene has the conserved Cdc25 function, we expected that overexpressing plants should show hypersensitivity towards genotoxic stress similar to the situation in human cells. 12,32 However, neither the gainnor the loss-of-function mutants showed a phenotype distinguishable from the wild type (Fig.…”