Marc J. Curtis

A shared DNA-damage-response pathway for induction of stem-cell death by UVB and by gamma irradiation.

Both UVB radiation and DNA-breaking agents were previously reported to kill Arabidopsis stem cells. We demonstrate that death induced by UVB or by ionizing radiation (IR) requires Suppressor of Gamma Response 1 (SOG1), a transcription factor already found to govern many responses to these agents in Arabidopsis. DNA-damage responses (DDRs) triggered primarily by replication-blocking photoadducts or double-strand-breaks thus converge to a shared programmed-cell-death (PCD) pathway. Both UVB- and IR-induced PCD also require functional DDR protein kinases. Employment of atr atm mutants (uniquely available in Arabidopsis) shows that either ATR (which recognizes ssDNA) or ATM (which recognizes DSBs) suffices for PCD induction by either agent. Thus, DNA damage made by UVB or by IR engenders both ATM-activating and ATR-activating structures. The elevated PCD in UVB-irradiated atr and atm mutants suggests that in wt plants ATR and/or ATM may activate both pathways that avert PCD and those that elicit it. The similar PCD levels induced by roughly 30,000 unrepaired photoadducts vs. 20 IR-induced DSBs indicate that DDR damage-tolerance activities in this model stem-cell niche are remarkably efficient.

Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3+/2 single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV32/2 homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I–V translocation. Spreads of meiosis I chromosomes in selfing AtREV3+/2 heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered.

Cooperative responses of DNA-damage-activated protein kinases ATR and ATM and DNA translesion polymerases to replication-blocking DNA damage in a stem-cell niche

Conserved DNA-damage responses (DDRs) efficiently cope with replication blocks and double-strand breaks (DSBs) in cultured eukaryotic cells; DDRs in tissues remain poorly understood. DDR-inactivating mutations lethal in animals are tolerated in Arabidopsis, whose root meristem provides a powerful stem-cell-niche model. We imaged UVB-induced death of specific meristem cells in single and double Arabidopsis mutants to elucidate cooperation among DNA translesion synthesis (TLS) polymerases (Polη, Polζ) and DNA-damage-activated protein kinases (ATR, ATM). Death was 100-fold higher in stem and progenitor (StPr) cells than in transiently amplifying cells. Quantitative analyses of dose-response plots showed that Polη and Polζ act redundantly to tolerate replication blocks and that Polζ-mediated TLS requires ATR. Deficient TLS resulted in ATM-signaled death, which first appeared 10-14h post-UVB. Although ssDNA downstream of blocks was likely cleaved into DSBs throughout S phase, death pathways appeared to initiate late in S. In atm mutants death appeared much later, likely signaled by a slow ATR-dependent pathway. To bypass replication blocks, tissues may use TLS rather than error-free pathways that could generate genomic aberrations. Dynamic balances among ATR and ATM death-avoidance and death-signaling functions determine how many DSB-burdened StPr cells are killed. Their replacement by less-burdened quiescent-center cells then restores growth homeostasis.