Double-Strand Break Repair in Plants Is Developmentally Regulated  

Abstract

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional b-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 3 10 28 ) being only 30% of the recombination rate at 2 dpg (9.14 3 10 28 ). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

“…We demonstrated that DNA damage and changes in HRF are not directly proportional to the received dose of ionizing radiation (Kovalchuk et al 2000). In the past, we also showed that plants exposed to stress early during development exhibit the highest increase in HRF (Boyko et al 2006b). Similarly to what was observed for changes in somatic HRF, increase in HRF in the progeny of stressed plants also depended on the severity of stress obtained in parental generation.…”

“…However, it should be noted that in the past, we prorated HRF according to the total number of genomes in the plant under analysis, thus calculating recombination rate (RR). We indeed found that in many cases, changes in HRF and RR in stressed plants were different (Boyko et al 2006b). Larger leaves often mean more cells, but this is not always the case.…”

“…Boyko et al (2006b) Fig. 7 The analysis of HRF, the size of recombination events, leaf size and HRF per leaf size in the progeny of plants exposed to different heat temperatures.…”

Role of AtPolζ, AtRev1, and AtPolη in UV Light-Induced Mutagenesis in Arabidopsis

“…In rice, several excision repair genes are expressed predominantly in shoot and root meristems but not in mature leaves (Kimura et al, 2004). It is known that homologous recombinations, an error-free repair pathway, take place more frequently at younger stages during plant development in Arabidopsis (Boyko et al, 2006). It is possible that higher plants employ error-free pathways to maintain an intact genome in undifferentiated tissue, whereas mutagenic bypass is utilized in differentiated tissues notwithstanding the risk of alterations to the genetic information.…”

Recognition and Repair Pathways of Damaged DNA in Higher Plants

“…Likewise, XRCC4 and Lig4 homologues have been described in plants, and functionally connected to NHEJ (West et al, 2000;Friesner and Britt, 2003;Kimura and Sakaguchi, 2006;Waterworth et al, 2010). In contrast to the error prone NHEJ pathway, HR is a more accurate repair mechanism that uses homologous DNA strands as templates for repair activities (Boyko et al, 2006a;Boyko et al, 2006b;Li and Ma, 2006;Osman et al, 2011). Several alternative pathways may exist that allow HR based repair of DSBs, however, good evidence is provided for at least two alternative pathways in plants.…”